Asp338 controls hydride transfer in Escherichia coli IMP dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the co ncomitant reduction of NAD(+). This reaction involves the formation of a co valent adduct with an active site Cys. This intermediate, E-XMP*, hydrolyze s to produce XMP. The mutation of Asp338 to Ala severely impairs the activi ty of Escherichia coli IMPDH, decreasing the value of k(cat) by 650-fold. N o V-D(m) or V-D(m) isotope effects are observed when 2-H-2-IMP is the subst rate for wild-type IMPDH. Values of V-D(m) = 2.6 and V-D/K-m (IMP) = 3.4 ar e observed for Asp338Ala. Moreover, while a burst of NADH production is obs erved for wild-type IMPDH, no burst is observed for Asp338Ala. These observ ations indicate that the mutation has decreased the rate of hydride transfe r by at least 5 x 10(3)-fold. in contrast, k(cat) for the hydrolysis of 2-c hloroinosine-5'-monophosphate is decreased by only 8-fold. in addition, the rate constant for inactivation by 6-chloropurine riboside 5'-monophosphate is increased by 3-fold. These observations suggest that the mutation has l ittle effect on the nucleophilicity of the active site Cys residue. These r esults are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2'-OH of MP, and the amide group of NA D(+) [Colby, T. D., Vanderveen, K., Strickler, M. D., Markham, G. D., and G oldstein, B. M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3531-3536].