ACTIVATION MECHANISM AND MODIFICATION KINETICS OF CHINESE-HAMSTER DIHYDROFOLATE-REDUCTASE BY P-CHLOROMERCURIBENZOATE

Substrate effects on the activation kinetics of Chinese hamster dihydr ofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the pre sence 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 C hinese hamster dihydrofolate reductase by pCMB shows single-phase kine tics, and that changes in the enzyme activity and tertiary structure p roceed 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 o f NADPH, the value of k(cat) for 7,8-dihydrofolate in the enzyme-catal ysed reaction increased four-fold on modification of Cys-6, accompanie d by a two-fold increase in K-m for the modified enzyme. The utilizati on of the binding energy of a group to increase k(cat) rather than red uce K-m implies that the full binding energy of the group is not reali zed in the formation of the enzyme-substrate complex, but is used to s tabilize the enzyme-transition-state complex.