Asp338 controls hydride transfer in Escherichia coli IMP dehydrogenase.
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-(2)H-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 x 10(3)-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].
Departments of Biochemistry and Biology, Brandeis University, Waltham, Massachusetts 02454, USA.
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Kerr
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Kuperwasser
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Hedstrom
D000478:Alkylation; D001224:Aspartic Acid; D002384:Catalysis; D020134:Catalytic Domain; D003903:Deuterium / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen / Q000378:metabolism*; D007168:IMP Dehydrogenase / Q000378:metabolism*; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation
D007291:Inosine Monophosphate; C086023:2-chloroinosine monophosphate; D001224:Aspartic Acid; C017193:deoxyinosine monophosphate; D006859:Hydrogen; D003903:Deuterium; D007168:IMP Dehydrogenase