Identification of an Arginine Residue in the Dual Coenzyme-Specific Glucose-6-Phosphate Dehydrogenase fromLeuconostoc mesenteroidesThat Plays a Key Role in Binding NADP+but Not NAD+

Glucose-6-phosphate dehydrogenase fromLeuconostoc mesenteroidescan utilize either NADP or NAD as coenzyme. The enzymeʹs three-dimensional structure has been solved (Rowlandet al.,1994,Structure2, 1073–1087) and shown to contain a conventional nucleotide binding domain. NADP+was modeled into the structure by superimposing the βαβ domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adamset al.,1994,Structure2, 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 theKmandKivalues 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 inL. mesenteroidesglucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes.