Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate.
Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme-substrate complex, but is used to stabilize the enzyme-transition-state complex.
Wu
J W
JW
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, Peoples' Republic of China.
Wang
Z X
ZX
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology*
D013439:Sulfhydryl Reagents; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D013762:Tetrahydrofolate Dehydrogenase