Abstract
IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+. This reaction involves the formation of a covalent adduct with an active site Cys. This intermediate, E-XMP*, hydrolyzes to produce XMP. The mutation of Asp338 to Ala severely impairs the activity of Escherichia coli IMPDH, decreasing the value of k cat by 650-fold. No D V m or D V/K m isotope effects are observed when 2-2H-IMP is the substrate for wild-type IMPDH. Values of D V m = 2.6 and D V/K m (IMP) = 3.4 are observed for Asp338Ala. Moreover, while a burst of NADH production is observed for wild-type IMPDH, no burst is observed for Asp338Ala. These observations indicate that the mutation has decreased the rate of hydride transfer by at least 5 × 103-fold. In contrast, k cat for the hydrolysis of 2-chloroinosine-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 little effect on the nucleophilicity of the active site Cys residue. These results are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2‘-OH of IMP, and the amide group of NAD+ [Colby, T. D., Vanderveen, K., Strickler, M. D., Markham, G. D., and Goldstein, B. M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3531−3536].