Thymidylate synthetase catalyzes an exchange of tritium of [5-3H]dUMP for protons of water in the absence of CH2-H4folate. The turnover number for this reaction is some 45,000-fold lower than that of dTMP formation and Km is 1.2 X 10(-5) M, similar to the dissociation constant of the enzyme-dUMP complex determined by equilibrium dialysis. The presence of 4 mM folate has no effect on Vmax but results in a decrease in the Km of dUMP to a value close to that in the normal enzymic reaction. The exchange reaction provides definitive evidence that the enzymic reaction involves attack of a nucleophile of the enzyme on the 6 position of dUMP to provide a 5,6-dihydro-dUMP intermediate which is covalently bound to the enzyme. Stereochemical considerations of the exchange reaction require proposal of a partial reaction which is not completely sterospecific or a complex reaction in which protons of water are handled with complete stereospecificity in a fashion similar to the one carbon unit of the normal enzymic reaction.
Vol. 67 groups or botti are necessary to the amylase activity. The extreme slowness of this irreversible inactivation, however, favors the idea that tyrosine rather than amino groups are involved. This conclusion was strengthened by further study. When the log fraction of the amylase activity which remained after inactivation by nitrous acid and subsequent reactivation with hydrogen sulfide was plotted against time, the irreversible inactivation was found to be first order with respect to the amylase. Furthermore, the constant obtained for this reaction was of the same order of magnitude as that obtained under similar conditions by Little and Caldwell315 for the formation of the azo compound with pure tyrosine. In addition, the same constant was obtained when the inactivation and reactivations were repeated with another concentration of the amylase.These results appear to rule out any appreciable influence either of deaminazation or of drastic oxidations and lead to the conclusion that free tyrosine is essential to the activity of betaamylase.
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