Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.
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.
Lee
W T
WT
Department of Biology, Syracuse University, New York 13244-1220.
Levy
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase; D008239:Lysine