Role of methionine-13 in the catalytic mechanism of 6-phosphogluconate dehydrogenase from sheep liver.
The crystal structure of sheep liver 6-phosphogluconate dehydrogenase (6PGDH) shows marked differences in the position of the nicotinamide mononucleotide (NMN) moiety of NADP(+) and NADPH (Adams, J. M., Grant, H. E., Gover, S., Naylor, C. E., and Phillips, C. (1994) Structure 2, 651-668). A methionine side chain (Met13) interacts with the si face of NADP(+) in the complex with the oxidized coenzyme, is likely to affect the binding mode of the nicotinamide ring of NADP(+), and may play a role in catalysis in the 6PGDH reaction. To check this possibility we performed site-directed mutagenesis, changing M13 to a number of residues including V, I, C, F, and Q. Mutant enzymes were characterized with respect to their kinetic parameters and primary deuterium isotope effects. All mutations resulted in a decrease in affinity of the enzyme for NADP(+), but not NADPH. In addition, the M13 to C (M13C), M13F, and M13Q mutant enzymes exhibited a decrease of at least an order of magnitude in V/E(t). The deuterium isotope effects on V and V/K(6PG) were decreased to about 1.2 for the M13F and M13C mutant enzymes, while they were increased to about 2.4 for the M13Q enzyme (a value of 1.8-1.9 is obtained for the wild-type enzyme). In at least three instances changes in the overall rate of the oxidative decarboxylation reaction relative to other steps along the reaction pathway were observed. Isotope effects indicate that the hydride transfer steps can become either more or less rate-determining dependent on the substitution. Data are consistent with a significant role of M13 in the orientation of the cofactor nicotinamide ring in the mechanism of 6PGDH, likely with respect to geometry and distance of the ring from C3 of 6PG.
Cervellati
Carlo
C
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, via Borsari 46, 44100 Ferrara, Italy.
Dallocchio
Franco
F
Bergamini
Carlo M
CM
Cook
Paul F
PF
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D041961:Deuterium Exchange Measurement; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008715:Methionine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D010649:Phenylalanine / Q000235:genetics; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012756:Sheep
D005973:Glutamine; D010649:Phenylalanine; D009249:NADP; D008715:Methionine; D010734:Phosphogluconate Dehydrogenase; D003545:Cysteine