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 (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2) .
Figure 2 HIF-1a in FH⁺ and FH⁻ 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?

Question

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 (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2) .

Figure 2 HIF-1a in FH⁺ and FH⁻ 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?

Quizplus · 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.

Passage Tumor Hypoxia Is a Marker of Resistance to Chemotherapy and and Radiation