pH dependence of enzyme reaction rates and deuterium isotope effects on the reduction of a new mechanism-based substrate by dihydrofolate reductase DHFR.
The enzyme kinetics of the reduction of the substrate 6,8-dimethylpterin by chicken and recombinant human dihydrofolate reductases (DHFRs) have been examined over the pH range 5.0-8.0 in the presence of NADPH or (4R)-[2H]NADPH (NADPD). The pH profiles of the catalytic constant (Vmax/[E]o or kcat) showed pH independence for chicken DHFR and little pH dependence for human DHFR. For both DHFRs, the pH profiles of the Michaelis constant (Km(substrate)) and the apparent second-order rate constant (Vmax/Km(substrate)[E]o or kcat/Km(substrate)) indicated that two ionizable groups, deduced to be the substrate and the conserved Glu carboxy group in the enzyme active site, should be ionized in their cationic and anionic forms, respectively, for formation of the enzyme-substrate complex and for catalysis. The pKa values of about 5.3 and 6.5 which were obtained from the pH profiles of Km(substrate) and kcat/Km(substrate) were assigned to the ionizations of the substrate and the enzyme carboxy group, respectively. Deuterium isotope effects on DV and d(V/K) were significant for both enzymes, approximately 3 for chicken DHFR and approximately 4 for recombinant human DHFR, and were pH independent. Thus, the rate-limiting step in the enzymic reduction of 6,8-dimethylpterin is hydride-ion transfer at acidic pHs as well as neutral pHs. The results demonstrate that, compared with dihydrofolate, 6,8-dimethylpterin is a superior substrate for mechanistic investigations as it allows direct study of the effects of both enzyme and substrate ionizations involved in the catalysis and also avoids the obscuration of the catalytic rate by product release.
Jeong
S S
SS
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Gready
J E
JE
D000818:Animals; D002384:Catalysis; D002645:Chickens; D003903:Deuterium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D011994:Recombinant Proteins; D009249:NADP; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase