Passage
NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs) , yielding the triphosphate form of each (NTPs and dNTPs) . dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast. Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP. Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication. It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP. The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate. PEI-cellulose separates nucleotides by charge (Figure 1) , and migration distance decreases with increasing negative charge. Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively.
Figure 1 TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP. (Note: Different nucleotides with the same phosphorylation level migrate different distances.) Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material. The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK. Products were separated by TLC and visualized (Figure 2) .
Figure 2 TLC analysis of dTDP phosphorylation with buffer only (Lane 1) , E75K TMPK (Lane 2) , or wild-type TMPK (Lane 3)
Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13) :2281-6.
-A Mg²⁺ ion in the active site of TMPK is directly involved in catalysis. Given this, Mg²⁺ most likely plays which of the following roles in TMPK-mediated reactions?

Question

Passage
NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs) , yielding the triphosphate form of each (NTPs and dNTPs) . dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast. Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP. Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication. It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP. The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate. PEI-cellulose separates nucleotides by charge (Figure 1) , and migration distance decreases with increasing negative charge. Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively.

Figure 1 TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP. (Note: Different nucleotides with the same phosphorylation level migrate different distances.) Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material. The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK. Products were separated by TLC and visualized (Figure 2) .

Figure 2 TLC analysis of dTDP phosphorylation with buffer only (Lane 1) , E75K TMPK (Lane 2) , or wild-type TMPK (Lane 3)
Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13) :2281-6.
-A Mg²⁺ ion in the active site of TMPK is directly involved in catalysis. Given this, Mg²⁺ most likely plays which of the following roles in TMPK-mediated reactions?

Quizplus · Accepted Answer

11ecba2d_0ee4_24a9_a3bf_3f9318892a63_MD0008_00 Catalysis is the acceleration of a reaction that occurs when a catalyst stabilizes the transition state between the reactant and the product. The stabilization of the transition state decreases the activation energy, or the energy necessary to initiate the reaction. Enzymes are biological catalysts that are primarily composed of amino acids but may require other components, known as cofactors, for catalysis. The question states that Mg²⁺ is directly involved in TMPK-mediated catalysis, so Mg²⁺ must be involved in stabilization of the transition state. Phosphate group transfer requires that ATP and dTMP form a high-energy-transition state in which their negative charges repel each other. The positively charged Mg²⁺ ion stabilizes this transition state by interacting with the negatively charged phosphates. As a result, the activation energy for the reaction is decreased in the presence of TMPK, and the reaction rate increases. (Choice A) The question states that Mg²⁺ is directly involved in catalysis. An enzyme may have a stable tertiary structure (conformational stability) but still be catalytically inactive if it lacks the necessary cofactors. (Choice B) Hydrolysis is the cleavage of a bond by adding water across it. TMPK catalyzes a phosphate transfer reaction, not a phosphate hydrolysis reaction. (Choice C) Mg²⁺ is directly involved in catalysis, so it must interact with the substrate directly. Preventing anions from competing for the active site could prevent inhibition but would not directly facilitate catalysis. Educational objective: Catalysis is the increase in a reaction rate that occurs when a catalyst decreases the activation energy of a reaction. Catalysts decrease activation energy by interacting with substrates to stabilize the transition state between reactant and product. Enzymes are biological catalysts, and some enzymes require cofactors such as metal ions for catalysis.

Passage NDPK Is an Enzyme in Budding Yeast That Uses ATP