THE ROLES OF CONSERVED CARBOXYLATE RESIDUES IN IMP DEHYDROGENASE AND IDENTIFICATION OF A TRANSITION-STATE ANALOG

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with t he concomitant reduction of NAD(+); the enzyme is activated by K+. Thi s reaction is the rate-limiting step in de novo guanine nucleotide bio synthesis. In order to identify functionally important residues in IMP DH, including those involved in substrate and K+ binding, we have muta ted 11 conserved Asp and Glu residues to Ala in Escherichia coli IMPDH . The values of k(cat), K-m, and K-i for GMP, XMP, mizoribine 5'-monop hosphate (MMP), and beta-methylene-tiazofurin adenine dinucleotide (TA D) were determined. Five of these mutations caused a significant chang e (greater than or equal to 10-fold) in one of these parameters. The A sp248 --> Ala mutation caused 100-fold decrease in the value of k(cat) and a 25-fold increase in the value of K-ii for TAD; these observatio ns suggest that Asp248 is in the NAD(+) binding site. The Asp338 --> A la mutation caused a 600-fold decrease in the value of k(cat), but onl y a 5-10-fold increase in the values of K-m for IMP and K-is for IMP a nalogs, suggesting that Asp338 may be involved in acid-base catalysis as well as IMP binding. The remaining three residues, Asp13, Asp50, an d Glu469, appear to be involved in Kf activation; these residues may b e ligands at one or more K+ binding sites. Interestingly, changes in t he values of K-i for MMP correlate with changes in k(cat)/KmKm of IMPD H, while no such correlation is observed for GMP, XMP, and TAD. This o bservation indicates that MMP is a transition state analog for the IMP DH reaction.