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 u tilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et at., 1994, Structure 2, 1073-108 7) and shown to contain a conventional nucleotide binding domain. NADP (+) was modeled into the structure by superimposing the beta alpha bet a 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 w as replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the K-m and K-i values for NADP(+) increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic consta nt was altered more than 6.5-fold. Changing this single amino acid lea ds to mutant glucose-6-phosphate dehydrogenases with coenzyme specific ities that favor NAD(+), whereas the wildtype enzyme prefers NADP(+) a s coenzyme. These results confirm that Arg-46 plays a key role in NADP (+) binding by contributing a positively charged planar residue that i nteracts primarily with the 2'-adenosine phosphate. The Arg residue co rresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrog enase is conserved in all glucose-6-phosphate dehydrogenases and, pres umably, plays the same role in all these enzymes. (C) 1996 Academic Pr ess, Inc.