Question 1

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -How is the enzymatic regulation of HIF-PHDs by fumarate and succinate best described?

Accepted Answer

11ecba2d_0dca_0280_a3bf_879778ba0541_MD0008_00 Figure 1 shows competitive inhibition of HIF-PHDs by succinate and fumarate on a Lineweaver-Burk plot, a graph based on a rearrangement of the Michaelis-Menten equation that is used to identify different types of enzyme inhibition. Lineweaver-Burk plots show the reciprocal of the substrate concentration (1/[S]) plotted against the reciprocal of the reaction velocity (1/V). Accordingly, the x-intercept is −1/K_M, the y-intercept is 1/V_max, and the slope is K_M/V_max. K_M, a measure of enzyme-substrate affinity, is defined as the substrate concentration at half of the maximum reaction velocity, or ½V_max. On these plots, increased K_M is observed as a right shift of the x-intercept (−1/K_M), and decreased V_max is shown as an upward shift of the y-intercept (1/V_max). Figure 1 shows that the substrate 2-OG is hydroxylated by HIF-PHD but that the inhibitors fumarate and succinate increase K_M (ie, right shift −1/K_M) while leaving V_max unaltered (ie, 1/V_max unchanged). Succinate and fumarate can be consequently classified as competitive inhibitors, molecules that bind reversibly to an enzyme's active site and block substrate access, causing an increase in K_M but no change in V_max. The unaltered V_max occurs because increased substrate concentration outcompetes the inhibitor, returning V_max to its original value. Succinate is a better competitive inhibitor as it results in a greater increase in K_M (greater right shift) than fumarate. Succinate occupies the HIF-PHD active site for longer periods than fumarate, effectively reducing the enzyme's affinity for 2-OG. (Choice A) Noncompetitive inhibitors allosterically bind both the enzyme and enzyme-substrate [ES] complex with the same affinity and decrease V_max but do not change K_M. (Choice B) Uncompetitive inhibitors, which allosterically bind [ES] only, reduce both V_max and K_M. (Choice C) Mixed inhibitors allosterically bind the enzyme or the [ES] complex with different binding affinities; this action is represented by a decrease in V_max and an increase or decrease in K_M. Educational objective: The x-intercept on a Lineweaver-Burk plot represents −1/K_M and the y-intercept represents 1/V_max. Competitive inhibitors bind reversibly to an enzyme's active site and block substrate access, causing an increase in K_M but no change in V_max.

Question 2

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -Fatty acids must be activated prior to degradation by a ligase enzyme. Which of the following reactions is catalyzed by a ligase?

Accepted Answer

11ecba2d_0dc8_a2ed_a3bf_5950a5840016_MD0008_00 The ligase class of enzymes catalyzes addition or synthesis reactions between two molecules by coupling them with the hydrolysis of energy-rich bonds such as those between ATP phosphate groups. Pyruvate carboxylase catalyzes the energy-requiring carboxylation of pyruvate to oxaloacetate, an intermediate for both amino acid synthesis and the citric acid cycle. Because pyruvate carboxylase "joined" pyruvate and CO₂ to form oxaloacetate, the enzyme is classified as a ligase. (Choice A) In this reaction, the transferase glycerol kinase moves a phosphate group from ATP to glycerol to form glycerol 3-phosphate. Transferases are used to shift functional groups from one molecule to another. (Choice B) A CO₂ molecule is removed from pyruvate by the lyase pyruvate decarboxylase. Lyases catalyze the cleavage of single molecules into two molecules (via breakage of C-C, C-O, C-N, and C-S bonds). These enzymes do not require water or energy from ATP and can form double bonds in their products. (Choice C) Alcohol dehydrogenase, an oxidoreductase, catalyzes the reduction of acetaldehyde to ethanol via concomitant oxidation of NADH to NAD⁺. Oxidoreductases catalyze reactions by facilitating the transfer of electrons between molecules with cofactors such as NAD⁺, NADP⁺, and FAD⁺. Educational objective: The ligase class of enzymes catalyzes addition or synthesis reactions between two molecules by coupling them with the hydrolysis of energy-rich bonds such as those between ATP phosphate groups.

Question 3

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -Based on the passage, culturing wild-type and FH⁻ cells in atmospheric oxygen conditions will lead to:

Accepted Answer

11ecba2d_0dcb_8921_a3bf_7d764b0b0ad2_MD0008_00 Figure 2 illustrates the effect of hypoxia and normoxia (normal/atmospheric oxygen levels) on the expression of the HIF-1α subunit in wild-type (FH⁺) and fumarase-deficient (FH⁻) cells. As expected, in hypoxic conditions both FH⁺ and FH⁻ cells express sufficient HIF-1α. In contrast, FH⁺ and FH⁻ cells grown in normoxic conditions lack HIF1-α. According to paragraph 1, HIF-1α should be less available under normal oxygen conditions because it is hydroxylated by active HIF-PHDs. Hydroxylation makes HIF-1α a target for ubiquitination, an ATP-dependent process in which ubiquitin molecules are added to a protein substrate. The proteasome then recognizes the ubiquitinated protein (eg, HIF-1α) and degrades it into small peptides via proteolysis (peptide bond cleavage). As a result, HIF-1α concentration is maximally decreased (or absent) under normoxia. In comparison, HIF-1α concentration is high during hypoxic conditions when HIF-PHDs are inactive. (Choice A) Normal oxygen levels induce HIF-1α degradation (not expression) by stimulating hydroxylation by active HIF-PHDs. (Choice B) Low (not normal) oxygen levels decrease the activity of HIF-PHDs, causing HIF-1α expression. (Choice D) Proteasomes will degrade more HIF-1α under normal atmospheric conditions because more will be ubiquitinated. Educational objective: Proteins are tagged for degradation in a process known as ubiquitination, which causes the proteasome to recognize and degrade the marked protein via proteolysis (peptide bond cleavage). This system is responsible for degrading damaged or unnecessary proteins/enzymes.

Question 4

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -In skeletal muscle cells, leptin resistance would lead to decreased concentrations of:

Accepted Answer

11ecba2d_0dc6_a717_a3bf_2b22eac4e2e3_MD0008_00 Leptin resistance or leptin insensitivity refers to an inability to increase fatty acid oxidation in a cell despite adequate leptin production and normal binding of leptin to cell surface receptors. The passage states that leptin generally activates AMPK, which then phosphorylates/inhibits ACC. If leptin is administered to skeletal muscles with leptin resistance, the exogenous leptin would still fail to activate the AMPK signaling pathway. AMPK would therefore remain inactive and unable to block ACC activity. As a result, ACC would remain uninhibited and continue to carboxylate free acetyl-CoA molecules to produce malonyl-CoA, which would increase in concentration (Choice C). The levels of acetyl-CoA would be reduced due to its continued conversion to malonyl-CoA, resulting in increased fatty acid synthesis and decreased β-oxidation. (Choices A and B) Leptin resistance leads to accumulation of fatty acid precursors (eg, malonyl-CoA), resulting in an increased concentration of triglycerides (composed of glycerol and fatty acids) and reduced oxidation of fatty acids. Educational objective: Leptin increases fatty acid oxidation and decreases fatty acid synthesis. In contrast, leptin-resistant cells cannot increase fatty acid oxidation despite adequate leptin signaling. As a result, AMPK signaling cannot be activated by leptin and leads to increased and uninhibited ACC activity, promoting fatty acid synthesis and reducing β-oxidation.

Question 5

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -The structure of Compound 1, which is found in adipocytes, is shown below. What would be the catabolic fate of this molecule?

Accepted Answer

11ecba2d_0dc3_24a4_a3bf_059caa951669_MD0008_00 Compound 1 is a triglyceride, which is composed of three fatty acid chains (part B) bonded to glycerol (part A) by an ester bond (gray line). When lipids must be degraded (catabolized) for fuel, triglycerides stored in adipocytes are first hydrolyzed by hormonally controlled lipases, yielding three fatty acids and one glycerol molecule. The fatty acids are released into the bloodstream and delivered to fuel-dependent tissues by albumin. Subsequently, the fatty acids are activated and enter the mitochondria for β-oxidation, which is their major catabolic pathway. Glycerol cannot be metabolized further by adipocytes; it is secreted into circulation and transported to the liver, where it is phosphorylated to glycerol-3-phosphate by glycerol kinase. Glycerol-3-phosphate is then converted to dihydroxyacetone phosphate (DHAP) by glycerol phosphate dehydrogenase. DHAP is isomerized to glycerladehyde-3-phosphate, an intermediate compound of glycolysis. (Choice B) Although glycerol (part A) can be incorporated into gluconeogenesis, it is an anabolic, not a catabolic pathway. Furthermore, free fatty acids (part B) are the final products of fatty acid synthesis, not the initial reagents. (Choice C) The reagents for fatty acid synthesis are acetyl-CoA and malonyl-CoA. Fatty acid chains with 16 carbon atoms (part B) do not enter fatty acid synthesis. Glycerol (part A) is not involved in fatty acid synthesis but is converted to DHAP for glycolysis. (Choice D) Glycogenolysis is a separate catabolic pathway that breaks down glycogen stored in the liver into glucose to increase ATP production. Free fatty acids (part B) are not glycogenolysis reagents, but glycerol (part A) can enter glycolysis. Educational objective: Triglycerides are storage lipid molecules that are broken down (catabolized) by lipases into glycerol and three fatty acids. Glycerol is converted into DHAP and incorporated into glycolysis or gluconeogenesis (anabolic pathway), depending on the cell's needs. The fatty acids diffuse into the mitochondria for β-oxidation, their major catabolic pathway.

Question 6

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -What best describes the action of PH on AMPK activity?

Accepted Answer

11ecba2d_0dc4_8435_a3bf_e7bbe19ea4e4_MD0008_00 In the study described in the passage, the control (baseline) was established as normal AMPK activity (at 100%). The activity of AMPK was assessed when PH was present or absent during leptin exposure (i.v. and i.h.p. at various time points). Muscles exposed to leptin only (no PH) showed a substantial increase in AMPK activity at i.v. 0.5 hr, i.v. 6 hr, and i.h.p. 1 hr. However, AMPK activity decreased in muscles exposed to both PH and leptin at i.v. 6 hr and i.h.p. 1 hr (due to faster drug delivery). This result signified that PH was blocking the leptin receptor and preventing AMPK activity from being increased by leptin, causing AMPK activity to return to a value close to baseline. PH can therefore be an antagonist, a compound that alters receptor-ligand binding affinity or binds a receptor directly to decrease the cell's response to the ligand. In addition, the increased amount of time taken for PH to lower AMPK activity (ie, inactive at 0.5 hr but active at i.v. 6 hr and 1 hr i.h.p) suggests that PH acts in a time-dependent manner. (Choices A and D) An agonist enhances or duplicates the effect of a ligand binding to a receptor. For PH to act as an agonist, it would have to bind to leptin-specific receptors and induce a biochemical response similar to that of leptin (ie, increase AMPK activity). (Choice B) AMPK activity was still increasing at i.v. 0.5 hr due to leptin exposure in the presence of PH. This increase implies that PH does not act immediately, which is supported by the fact that AMPK activity returned to a value close to baseline only with leptin (plus PH) at i.v. 6 hr. Educational objective: An agonist enhances or duplicates the effect of a ligand binding to a receptor. Antagonists alter receptor-ligand binding affinity or bind directly to a receptor to decrease the cell's response to the ligand. The effect of agonists or antagonists can be time-dependent (ie, may not be immediate).

Question 7

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens. One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system. Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs. Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1 Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins. To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment. The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2 Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors. Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3 Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -The figure below shows the active site of NSP4 with three residues that are essential for catalysis. If site-directed mutagenesis was used to create a missense mutation at His⁵⁷, this would likely result in which of the following changes regarding the enzyme-catalyzed degradation of the viral peptide depicted in the active site?The time required for the reaction to achieve equilibrium would increase.The equilibrium constant K_eq of the reaction would decrease.The enzyme-substrate dissociation constant K_d would increase.

Accepted Answer

11ecba2d_0dd4_d803_a3bf_2d139ac6e7de_MD0008_00 Enzymes are biological catalysts that lower the activation energy of a reaction, allowing the reaction to proceed at a faster rate. Substrate molecules bind to the enzyme's active site, where catalytic activity occurs. The enzyme and substrate undergo conformational changes that make the transition state more stable and favorable, resulting in lower activation energy. Enzyme function is sensitive to changes in pH and temperature as well as deleterious mutations, particularly those involving the active site. A missense mutation at the histidine in position 57 of the NSP4 sequence (ie, His⁵⁷) would result in a different amino acid at this site and alter the interaction between the viral peptide (substrate) and the NSP4 active site. This mutation would be expected to decrease the binding affinity of the enzyme for its substrate, resulting in an increased dissociation constant K_d for the enzyme-substrate complex (Number III). As a result, the mutation would interfere with normal enzymatic function and lead to a decreased reaction rate (ie, increasing the time the reaction takes to reach equilibrium) (Number I). (Number II) Enzymes do not alter the free energy change ΔG or the equilibrium constant K_eq of a reaction. The overall ΔG depends only on the energy difference between products and reactants (ΔG = G_products − G_reactants), and can be visualized in a free energy diagram. The equilibrium constant K_eq of a reaction is the ratio of products to reactants for a given reaction at equilibrium at a specific temperature and is related to ΔG (ie, ΔG = −RT ln K_eq). Although a functional enzyme would decrease the time taken for the reaction to reach equilibrium (ie, ΔG = 0 for the reaction) by increasing the reaction rate, it would not alter K_eq. Educational objective: Enzymes do not alter the free energy change ΔG or the equilibrium constant K_eq of a reaction but do decrease the time taken for the reaction to reach equilibrium by increasing the reaction rate. Deleterious mutations involving the enzyme's active site are likely to alter the enzyme's affinity for its substrate (increase K_d) and interfere with enzymatic function (decreased reaction rate).

Question 8

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -The addition of AMPK phosphatases to skeletal muscles exposed to leptin would result in which of the following?

Accepted Answer

11ecba2d_0dc5_95a6_a3bf_996cf57de028_MD0008_00 Phosphatases remove phosphate groups from enzymes/substrates (ie, dephosphorylation), whereas kinases add phosphate groups (ie, phosphorylation). In this scenario, AMPK phosphatases in leptin-exposed muscles would dephosphorylate AMPK and thereby inactivate it. Inactive AMPK would be unable to inhibit (phosphorylate) ACC, which carries out the first and rate-limiting step of fatty acid synthesis by carboxylating acetyl-CoA to malonyl-CoA. As a result, unphosphorylated ACC would promote fatty acid synthesis, preventing β-oxidation (Choice D). Fatty acid synthase carries out the main reactions of fatty acid synthesis and contains an acyl carrier protein (ACP) responsible for transferring acyl intermediates. Acetyl-CoA and malonyl-CoA are activated to acetyl-ACP and malonyl-ACP, which are condensed into acetoacetyl-ACP. Subsequently, acetoacetyl-ACP is reduced, dehydrated, and further reduced to generate butyryl-ACP. The coenzyme NADPH is used for both reductive steps. The reactions are repeated until a 16-carbon fatty acid (palmitate) is formed via addition of two carbon atoms per cycle. (Choice A) In β-oxidation, a carnitine transporter moves 14-16 carbon fatty acids from the cytosol into the mitochondria. However, β-oxidation is inhibited in this setting. Instead, fatty acid synthesis requires shuttling of citrate from the mitochondria into the cytosol to yield the acetyl-CoA precursor. (Choice B) NAD⁺ and FAD are reduced to NADH and FADH₂ (electrons gained) in β-oxidation. However, fatty acid synthesis requires NADPH (as an electron donor) to reduce the acyl groups added to the fatty acid. Educational objective: In the cytosol, ACC catalyzes the first (rate-limiting) step of fatty acid synthesis by carboxylating acetyl-CoA to malonyl-CoA. Fatty acid synthase then carries out the main reactions of fatty acid synthesis. This enzyme contains an acyl carrier protein, which transfers acyl intermediates.

Question 9

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2. Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -During cis-complex disassembly, what consequence of NSF function is most likely responsible for destabilization of the cis-complex?

Accepted Answer

11ecba2d_0dbd_0a19_a3bf_bb8d5dccb503_MD0008_00 Typically, hydrophilic amino acids (polar and charged) are found on the outside of proteins, where they interact favorably with water molecules. Hydrophobic amino acids are usually shielded from the solvation layer in the protein interior or found at protein-protein interfaces. This arrangement is entropically favorable, as water molecules cannot form hydrogen bonds with hydrophobic residues, and therefore must organize around these residues. Leucine is hydrophobic, and leucine zipper layers act as a watertight seal around the zero ionic layer, in which a positively charged, basic arginine (R) residue interacts with polar glutamine (Q) residues at the interface of the complexed proteins. NSF destabilizes the cis-complex by breaking the leucine zipper seal (unzipping). Without the watertight seal provided by the leucine zipper, the charged and polar residues of the zero ionic layer are exposed to water. Because water is polar, exposure of the charged and polar residues results in preferential binding to water and resultant destabilization of the cis-complex. (Choice B) The charges on residues in the zero ionic layer do not change, so no additional repulsive forces are added by breaking the leucine zipper. (Choice C) A leucine zipper is a common protein motif composed of two amphipathic α-helices, meaning they possess both hydrophilic and hydrophobic residues. Unzipping would not change the residues, and therefore would not change the amphipathic qualities of the zipper. (Choice D) Release of v-SNARE proteins would not occur until after cis-complex disassembly. Educational objective: Hydrophilic amino acids are typically found on the outside of proteins whereas hydrophobic amino acids exist in the protein interior and protein-protein interfaces. When polar or charged amino acids are exposed to water, they will preferentially form hydrogen bonds with water molecules.

Question 10

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -During hypoxia, skeletal muscles have been shown to accumulate succinate and ADP molecules. Which of the following explains this result?

Accepted Answer

11ecba2d_0dcf_cee8_a3bf_c9267fe6e6ba_MD0008_00 The accumulation of succinate and ADP during hypoxia would occur because anaerobic conditions prevent the normal regeneration of the NAD⁺ and FAD needed for progression of the citric acid cycle. This cycle involves reactions that occur within the mitochondrial matrix and perform aerobic processing (oxidation) of pyruvate to generate CO₂ and ATP, the cell's energy currency. The dehydrogenase enzymes in this cycle transfer hydride ions to an electron acceptor (eg, NAD⁺ or FAD), creating the high-energy electron carriers NADH and FADH₂. NADH and FADH₂ are then shuttled to the electron transport chain (ETC) in the inner mitochondrial membrane where proteins transfer the released electrons to oxygen, the final electron acceptor. NAD⁺ and FAD are also regenerated, and the electron movement between ETC proteins generates a proton motive force that drives ATP synthase to phosphorylate ADP to ATP. During hypoxia, less oxygen is available to accept electrons from the citric acid cycle; therefore, NAD⁺ and FAD are not regenerated in the mitochondria. Consequently, ADP concentrations increase (due to the absent proton motive force) as ATP production is significantly reduced. In addition, there is an accumulation of succinate and other citric acid cycle intermediates that require NAD⁺ or FAD for their oxidation reactions. (Choice B) Succinate dehydrogenase remains fully functional during hypoxia, so its k_cat is unchanged. However, because FAD is a substrate of succinate dehydrogenase, hypoxia-induced depletion of FAD causes a decrease in the reaction rate. (Choice C) Fumarate is not converted back to succinate in ATP-rich conditions. Instead, excess citric acid cycle intermediates such as fumarate are used as amino acid precursor molecules. (Choice D) High concentrations of NADH inhibit the α-ketoglutarate dehydrogenase that generates succinyl-CoA from α-ketoglutarate. As a result, succinyl-CoA production is reduced, not stimulated. Educational objective: The citric acid cycle uses oxidation reactions to convert NAD⁺ and FAD into NADH and FADH₂, respectively. The movement of electrons from these high-energy electron carriers to electron transport chain (ETC) proteins drives phosphorylation of ADP to ATP. Regeneration of NAD⁺ and FAD in the ETC is only possible when oxygen is available to act as a final electron acceptor.

Question 11

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -Which amino acid structure would most likely be hydroxylated by HIF-PHDs?

Accepted Answer

11ecba2d_0dd1_cabd_a3bf_db057a84e654_MD0008_00 Hydroxylation reactions are involved in the oxygen-induced degradation of organic compounds and entail the addition of a hydroxyl group (-OH), usually in place of a hydrogen (H) atom. In the passage, HIF-PHD hydroxylates HIF at aliphatic residues. Aliphatic amino acids are nonpolar and hydrophobic ("water-fearing") because their R-groups are composed mostly of carbon atoms that can have single, branched, or cyclic (nonaromatic) carbon backbones. Hydrophobicity increases as the number of carbon atoms in the side chain increases. For this reason, glycine, which is nonpolar but has an H atom as its R-group, is often excluded from the list of aliphatic amino acids. However, methionine is often included due to the inert nature of its sulfur atom. Of the choices listed, proline (Choice A) is the only amino acid with an aliphatic side chain; therefore, proline is most susceptible to hydroxylation by HIF-PHDs. (Choice B) Histidine is classified as a basic amino acid, and its R-group can be positively charged (N-atom protonated) or uncharged at neutral pH. Histidine can also act as a nucleophile by donating its electrons to electron-deficient atoms. (Choice C) Glutamic acid is an acidic amino acid that already has a carboxylic acid R-group. (Choice D) Phenylalanine is a nonpolar amino acid but cannot be classified as aliphatic due to the aromatic ring in its R-group. Educational objective: Hydroxylation reactions replace hydrogen atoms with hydroxyl (-OH) groups and can occur in amino acids with aliphatic R-groups. Aliphatic amino acids are nonpolar, hydrophobic molecules with R-groups consisting of chains of C-C bonds that are single, branched, or in nonaromatic rings.

Question 12

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance. When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization. One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles. To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes. Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice. Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1 AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1 ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -What are the most likely role and effect of citrate on ACC activity?

Accepted Answer

11ecba2d_0dc7_9178_a3bf_0ff9f2482342_MD0008_00 Allosteric effectors regulate enzymatic activity by binding at sites other than the active site and causing conformational changes in the enzyme. As a result, enzymatic activity is either decreased (ie, inhibited) or increased (ie, activated). Table 1 shows that regardless of saline or leptin administration, ACC activity increases when citrate (2 nM) is added. Therefore, citrate is most likely an allosteric effector that activates ACC. Note that any increase in enzyme activity is considered activation, even if the enzyme had some non-zero activity in the absence of the allosteric effector. (Choices A and B) Cofactors, including metal ions and coenzymes (small organic compounds), bind to the enzyme's active site and are required for enzymatic activity. The ACC enzyme is active in the absence of citrate, so citrate is neither a coenzyme nor a cofactor. (Choice D) Inhibitors decrease enzyme activity. Table 1 indicates that the addition of citrate (2 mM) corresponds to increased ACC activity. Educational objective: Allosteric effectors regulate enzymatic activity by binding at sites other than the active site and causing conformational changes in the enzyme. As a result, enzymatic activity is either inhibited or activated (ie, increased).

Question 13

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -The peptide substrate (PS) binds HIF-PHDs outside of the active site. PS binds to both HIF-PHD and HIF-PHD-2-OG complexes with equal affinity. According to the passage, which graph would represent the effect of PS on HIF-PHD activity?

Accepted Answer

11ecba2d_0dce_2137_a3bf_39b721bb93a1_MD0008_00 Michaelis-Menten graphs plot initial reaction velocities V₀ against substrate concentration [S] and can be used to determine V_max and K_M. As [S] increases, V₀ also increases up to a maximum reaction velocity V_max, resulting in a hyperbolic curve. The Michaelis constant K_M is defined as the substrate concentration where half of V_max is achieved (ie, V_max/2) or where half of the enzyme's active sites are full. K_M quantitatively represents the enzyme's affinity for its substrate. Changes in K_M and V_max can be used to identify types of enzyme inhibition. In this scenario, PS binds both HIF-PHD allosterically (outside the active site) and HIF-PHD-2-OG with equal affinity. By definition, PS is considered a noncompetitive inhibitor, a molecule that binds both the enzyme and the enzyme-substrate complex (ES) with equal affinity. Noncompetitive inhibitors slow the reaction rate and decrease V_max; however, they do not alter the affinity of the enzyme for its substrate, resulting in an unchanged K_M. Accordingly, in the fourth plot of reaction velocity V₀ with increasing 2-OG substrate, the second curve shows that addition of PS decreases V_max but does not change the K_M compared to the first curve. (Choice A) This graph shows uncompetitive inhibition, which decreases both V_max and K_M by the same factor, as these inhibitors only bind to [ES]. However, the PS inhibitor binds both the enzyme and [ES]. (Choice B) This graph shows an increase in K_M but no change in V_max when PS is added. This represents the action of competitive inhibitors that bind to the active site and prevent substrate binding, an effect that can be overcome by adding more substrate, resulting in an unchanged V_max. (Choice C) The type of mixed inhibition displayed in this graph increases K_m while decreasing V_max. Mixed inhibitors generally decrease V_max and have different binding affinities for the enzyme and [ES] at allosteric sites; as a result, they can increase or decrease K_M. Educational objective: Michaelis-Menten graphs plot the initial reaction velocity against substrate concentrations and can be used to determine K_M and V_max. Noncompetitive inhibitors allosterically bind the enzyme and enzyme-substrate complex with equal affinity. These inhibitors decrease V_max but do not alter K_M.

Question 14

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens. One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system. Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs. Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1 Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins. To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment. The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2 Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors. Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3 Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -What is the activation energy for the uncatalyzed reaction in Figure 2?

Accepted Answer

11ecba2d_0dd3_5161_a3bf_f1f69629afaa_MD0008_00 In a free energy diagram, the x-axis represents the progress of a reaction (reaction coordinate) and the y-axis depicts the Gibbs free energy G of the system. Reactants and products are separated by an energy barrier known as the energy of activation E_a, defined as the energy difference between the transition state (‡) and reactants in a reaction (ie, E_a = E_‡ − E_reactants). The transition state of a reaction is the intermediate point of greatest energy where bonds break and form as the product is generated. For a reaction to proceed, the reactants must possess sufficient energy to overcome E_a. The height of the E_a barrier determines the reaction rate (ie, the amount of product formed per unit time), and the greater the E_a, the slower the reaction. Uncatalyzed reactions have a greater E_a than catalyzed reactions, and therefore are slower. Without a catalyst, only a few molecules have sufficient energy to overcome the E_a barrier and achieve the transition state (‡_uncatalyzed). However, the addition of a catalyst increases the rate of a reaction by lowering the E_a barrier, allowing more reactants to achieve the transition state and form the product faster. Figure 2 shows the reaction pathway of an uncatalyzed reaction compared to a reaction catalyzed by NSP4. Using the given formula for the uncatalyzed reaction, E_a is calculated as 24 − 4 = 20 kcal/mol. (Choice A) The catalyzed reaction shows that only 8 kcal/mol (E_a1) and 4 kcal/mol (E_a2) are needed to achieve the first (‡₁) and second (‡₂) transition states, respectively. The sum of these energies is 12 kcal/mol. (Choice C) The total energy of the transition state for the uncatalyzed reaction is 24 kcal/mol. (Choice D) This value represents E_a of the reverse reaction, which is the energy difference between the transition state and the products (ie, E_a,reverse = E_‡ − E_products), and is equivalent to 28 kcal/mol. Educational objective: The activation energy E_a is the minimum energy required for reactants to reach the transition state and initiate a reaction (given by E_a = E_‡ − E_reactants). Uncatalyzed reactions have greater E_a than catalyzed reactions, and therefore are slower. Catalysts allow the reaction to proceed at a faster rate by lowering the activation energy.

Question 15

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation. The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization. Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs. These hydroxylated residues serve as markers for the ubiquitin-proteasome system. Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs. Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1 Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FHS1U1P1+S1S1P0) and fumarate-deficient (FHS1U1P1−S1S1P0) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2 HIF-1a in FHS1U1P1+S1S1P0 and FHS1U1P1−S1S1P0 cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -Why did researchers test for the α-tubulin protein along with HIF-1α?

Accepted Answer

11ecba2d_0dcc_9a92_a3bf_2944c7e31a1a_MD0008_00 Depicted in Figure 2, western blotting uses luminescent antibodies to identify proteins separated by gel electrophoresis. The protein concentrations detected are represented by bands, which may vary in width/intensity (corresponding to greater protein abundance). Proteins that are ubiquitously expressed (ie, they have consistent concentration levels across all cell/tissue types) are used as loading controls to show that all lanes in the gel had the same amount of sample loaded into them. Properly loaded gels have the same amount of loading control in every lane. The most common loading controls are proteins necessary for baseline cellular function that are transcribed from housekeeping genes (eg, α- and β-tubulin proteins are structural/mobile cytoskeleton components). Based on this fact, α-tubulin was used as a loading control as it can be detected at consistent levels in all cell types and has a molecular weight different from HIF-1α. The consistent expression of α-tubulin in the gel under all conditions confirms that differences in HIF-1α expression were not a result of experimental errors in protein loading or detection. (Choice A) An increase in fumarate would not affect α-tubulin levels because loading controls are always present and remain unaffected by changes in experimental conditions. (Choice C) A negative control is a standard experimental condition in which no response is expected (eg, using cells unable to express HIF-1α). However, α-tubulin is always expressed, and its presence does not confirm/predict the absence of HIF-1α. (Choice D) A positive control is one in which the expected result is always observed (eg, using cells already known to always produce HIF-1α). α-Tubulin is not a positive control because it does not confirm/predict the presence of HIF-1α, the expected result under hypoxic conditions. Educational objective: Loading controls normalize protein detection and ensure that protein loading is standardized across the gel. Proteins used as loading controls tend to be ubiquitously expressed and have consistent concentrations across all cell/tissue types. Housekeeping genes are the most common loading controls.

Question 16

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens. One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system. Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs. Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1 Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins. To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment. The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2 Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors. Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3 Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -Considering that NSPs are proteases, which of the following statements best describes the reaction catalyzed by NSPs?

Accepted Answer

11ecba2d_0dd6_3794_a3bf_45091decc2bc_MD0008_00 Peptides are linear sequences of amino acids linked by amide bonds (-CO-NH-) that join the α-carboxyl group of one amino acid to the α-amino group of another amino acid. Proteins that have completed their biological purpose must be degraded by proteases so that their constituent amino acids can be reused to generate new peptides. To cleave an amide bond during protein degradation, a water molecule (H₂O) must be added using acids (H⁺) or bases (OH⁻) in a reaction known as amide hydrolysis. Figure 1 shows the acylation process (R-C=O addition) of amide hydrolysis by NSP4, which cleaves the amide bond of the viral peptide by adding a hydrogen atom to the nitrogen. The hydrogen atom displaces the C-N single bond when electrons from the oxygen on the carbonyl carbon reform the double bond. The acyl group (R₁-C=O) attached to NSP4 is subsequently converted into carboxylic acid when electrons from an OH⁻ group attack the electrophilic (electron-attracting) carbonyl carbon. (Choices A, C and D) Hydrolysis reactions only involve consumption of water to break bonds. They do not involve water production, which occurs in condensation reactions such as peptide bond formation. Educational objective: Proteins can be degraded by protease enzymes. During peptide hydrolysis, a water molecule is used to cleave the C-N bond in the amide linkage using an acid (H⁺) or base (OH⁻). In contrast, the reverse of amide hydrolysis is amide bond condensation, which involves the formation of a water molecule from two amino acids to form a larger peptide.

Question 17

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2. Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Wild-type syntaxin (Synt-WT) and mutant syntaxins containing a deletion of the N-terminal sequence (SyntΔA) or C-terminal sequence (SyntΔB) were separated and incubated with α-SNAP. After washing away unbound molecules, which of the following is the most likely result of protein immunoblotting for α-SNAP?

Accepted Answer

11ecba2d_0dc1_9e00_a3bf_87c441b24ce1_MD0008_00 Western blot (protein immunoblotting) is a protein separation and identification technique that uses labeled antibodies to detect specific proteins after gel electrophoresis. In this example, the antibody is designed to bind α-SNAP. Therefore, after unbound α-SNAP is washed away, protein samples will be visualized on western blot in proportion to α-SNAP binding. The passage indicates that α-SNAP binds syntaxin at its N-terminal domain. The mutant syntaxin SyntΔA described in the question lacks the N-terminal domain and is unlikely to bind α-SNAP in large quantities, leading to little α-SNAP-bound syntaxin for this mutation. In contrast, deletion of the C-terminal domain (SyntΔB) is not expected to affect α-SNAP binding compared to binding of the WT protein. Therefore, α-SNAP in the WT protein and SyntΔB samples will be visualized as large bands whereas less α-SNAP will be visualized in the SyntΔA sample. (Choice B) α-SNAP poorly binds the mutant syntaxin SyntΔA. The quantity (band intensity) of α-SNAP-bound SyntΔA should be less than WT and SyntΔB. (Choice C) According to the passage, α-SNAP binds syntaxin's N-terminal domain. Modification of the C-terminal domain in SyntΔB should not impact α-SNAP binding, so the SyntΔB lane should look similar to the WT lane. (Choice D) Synt-WT binds α-SNAP effectively, so the western blot should show a full band in the WT lane. Educational objective: Western blot (protein immunoblotting) uses antibodies to detect specific proteins. Greater quantities of the protein of interest will manifest as larger bands on the resultant blot.

Question 18

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2. Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Which of the following amino acid substitutions is most likely to interrupt the interactions of the zero ionic layer?

Accepted Answer

11ecba2d_0dbe_1b8a_a3bf_4363725446af_MD0008_00 Amino acid substitutions are denoted by the original amino acid followed by the amino acid position and the substituting amino acid. Amino acid substitutions that alter charge are likely to destabilize protein interactions. In this example, Q53R denotes the replacement of glutamine with arginine at position 53 on SNAP-25 (Sn1). As the passage suggests, the zero ionic layer is stabilized by attractive forces between the carbonyl groups (partial negative charge) on glutamine residues and the positively charged arginine on synaptobrevin. Substitution of glutamine with arginine results in a repulsive positive-positive ion interaction between the two arginine residues, destabilizing the interaction between the proteins in the complex. (Choice A) R56K denotes the substitution of arginine with lysine on synaptobrevin. Because both amino acids are positively charged, this substitution would be expected to have a relatively small effect on protein-protein interactions. (Choice C) E224D denotes the substitution of glutamate at position 224 on syntaxin with aspartate. According to Table 1, it is residue 226 rather than 224 that is in the zero ionic layer. In addition, because glutamate and aspartate are both negatively charged, this substitution is unlikely to significantly alter the properties of the protein. (Choice D) Q174N denotes the substitution of glutamine with asparagine on SNAP-25 (Sn2). Asparagine is similar in structure to glutamine, and will therefore most likely interact similarly within the zero ionic layer. Educational objective: Amino acid substitutions are denoted by the original amino acid, the amino acid position, and the substituting amino acid. Amino acid substitutions that alter charge are likely to destabilize protein interactions, particularly if they result in repulsive forces between similarly charged amino acids.

Question 19

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens. One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system. Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs. Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1 Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins. To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment. The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2 Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors. Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3 Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -Which of the following is the most accurate statement regarding NSP4 binding of viral proteins?

Accepted Answer

11ecba2d_0dd7_7015_a3bf_cdd8f7d3202b_MD0008_00 Two competing theories exist regarding enzyme-substrate interaction, the lock and key theory and the induced fit model. The lock and key theory proposes that an enzyme's active site is already in the proper structural conformation to allow a substrate to bind readily and form an active enzyme-substrate (ES) complex. This theory proposes that no conformational changes are necessary for catalysis to occur. The induced fit model states that a conformational change is induced when the substrate binds the active site, resulting in the formation of a functional ES complex that is said to be in the induced form. Enzymes are highly specific for their substrates, and only the correct substrate can induce the required complementary change in active site conformation. Shifting an enzyme's tertiary or quaternary structure to alter the shape of the active site requires energy. The passage states that NSP4 alters its tertiary structure upon substrate binding, so NSP4 most likely undergoes a conformational change that requires energy upon substrate binding. (Choice B) Quaternary structure is only present in multimeric proteins that have multiple subunits. The passage states that NSP4 is a monomer (one subunit), so it does not have quaternary structure. (Choice C) When the correct substrate enters and is bound by an enzyme's active site, heat is released (not consumed) from the formation of weak bonds (hydrogen, hydrophobic, ionic bonds) between the two molecules. (Choice D) Cofactors are molecules required for enzyme catalysis. Enzymes that require cofactors for activity are called apoenzymes when the cofactor is absent whereas those containing cofactors are known as holoenzymes. The passage does not indicate that NSP4 needs a cofactor for activity. Educational objective: The lock and key model proposes that the active site of an enzyme is already in the correct conformation to bind to a substrate. In contrast, the induced fit theory states that the initial binding of a substrate causes the active site to undergo a conformational change that complements the structure of the substrate.

Question 20

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2. Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Which of the following statements regarding the binding of α-SNAP to syntaxin is best supported by the results shown in Figure 2?

Accepted Answer

11ecba2d_0dbf_2cfb_a3bf_dfd50e0623ee_MD0008_00 Substrate binding curves relate the number of bound substrate molecules (percent saturation) to substrate concentration. Initially, substrate-protein binding is approximately linear. When proteins reach saturation, all binding sites are occupied; this condition can be observed as the leveling off of the binding curve at the binding limit, which is dependent on the amount of protein present and the number of binding sites. At this binding limit, addition of more substrate does not result in increased binding. Figure 1 shows that when synaptobrevin is present, increasing α-SNAP beyond 2 μM does not result in increased binding, suggesting that all binding sites are saturated. Conversely, when synaptobrevin is absent, α-SNAP binding to syntaxin is reduced, and the amount of α-SNAP bound to syntaxin increases linearly with increasing α-SNAP concentration over the range tested, suggesting that binding has not approached saturation. (Choice A) The data show that α-SNAP binds to syntaxin to different extents in both the presence and absence of synaptobrevin. (Choice B) The amount of α-SNAP bound to syntaxin increases when synaptobrevin is present. This behavior suggests a higher binding affinity in the presence of synaptobrevin. (Choice D) The dissociation constant K_d is an equilibrium constant for the dissociation of bound molecules into two or more unbound molecules. Increased K_d reflects decreased binding affinity. The data in Figure 2 suggest the opposite, that synaptobrevin interacting with syntaxin increases α-SNAP binding affinity and decreases the K_d for α-SNAP dissociation. Educational objective: On substrate binding curves, substrate-protein binding is approximately linear at low substrate levels. When proteins reach saturation, all binding sites are occupied and binding curves level off because the further addition of substrate will not increase binding.

Exam 2: Biochemistry