Lysine‐21 of Leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase participates in substrate binding through charge–charge interaction

Abstract

Leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6‐phosphate dehydrogenase gene. Lys‐21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5′‐phosphate and pyridoxal 5′‐diphosphate‐5′‐adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6‐phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558–5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys‐21. Both mutant enzymes showed increased Km‐values for glucose 6‐phosphate compared to wild‐type enzyme: 1.4‐fold (NAD‐linked reaction) and 2.1‐fold (NADP‐linked reaction) for the K21R enzyme, and 36‐fold (NAD‐linked reaction) and 53‐fold (NADP‐linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5‐ and 3.2‐fold, compared to the wild‐type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD‐ and NADP‐linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD‐linked reaction by 26% and decreased by 30% in the NADP‐linked reaction from the values for the wild‐type enzyme. The data are consistent with Lys‐21 participating in the binding of the phosphate group of the substrate to the enzyme via charge–charge interaction. Copyright © 1992 The Protein Society