Glycerol-3-phosphate dehydrogenase (GPDH) serves as a catalyst for the reversible reaction of dihydroxyacetone phosphate (DHAP) and NADH to form L-glycerol 3-phosphate (L-G3P) and NAD+. We are interested in defining the catalytic roles of the K120 and K204 side chains, which are positioned at the active site to interact with the carbonyl group of DHAP (Figure 1). We previously characterized the effect of the K120A substitution on activity. We report here data for catalysis by K204A and K120A/K204A variants, which demonstrate an important catalytic role for these two lysine residues. We have used polyhistidine affinity tags (HisTAGs) to simplify the purification of wildtype and variant forms of GPDH. We report that the presence of the HisTAG has only a small effect on the activity of wildtype and variant enzymes towards catalysis of reduction of DHAP by NADH, but results in 400-fold enhancement in the activity for K204A variant- compared to wildtype enzyme-catalyzed reduction of the truncated substrate glycolaldehyde. There is good evidence that wildtype hlGPDH has evolved to optimize the utilization of phosphodianion binding interactions in stabilization of the transition state for reduction of DHAP by NADH. We summarize the enzyme modifications that are observed to stabilize a second transition state for reaction of small neutral substrates, where there are minimal stabilizing interactions with added phosphite dianion.

Human glycerol 3–phosphate dehydrogenase: Effect of polyhistidine tag on enzyme function and activity

ACS Spring Meeting 2021

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