Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+.
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes.
Levy
H R
HR
Department of Biology, Syracuse University, New York 13244-1220, USA. hrlevy@mailbox.syr.edu
Vought
V E
VE
Yin
X
X
Adams
M J
MJ
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D007934:Leuconostoc / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D001120:Arginine; D005954:Glucosephosphate Dehydrogenase