The roles of conserved carboxylate residues in IMP dehydrogenase and identification of a transition state analog

Abstract

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+; the enzyme is activated by K+. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. In order to identify functionally important residues in IMPDH, including those involved in substrate and K+ binding, we have mutated 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'-monophosphate (MMP), and β-methylene- tiazofurin adenine dinucleotide (TAD) were determined. Five of these mutations caused a significant change (≤ 10-fold) in one of these parameters. The Asp248 → 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 observations suggest that Asp248 is in the NAD+ binding site. The Asp338 → Ala mutation caused a 600-fold decrease in the value of k(cat), but only a 5- 10-fold increase in the values of K(m) for IMP and K(is) for IMP analogs, suggesting that Asp338 may be involved in acid-base catalysis as well as IMP binding. The remaining three residues, Asp13, Asp50, and Glu469, appear to be involved in K+ activation; these residues may be ligands at one or more K+ binding sites. Interestingly, changes in the values of K(i) for MMP correlate with changes in k(cat)/K(m)K(m) of IMPDH, while no such correlation is observed for GMP, XMP, and TAD. This observation indicates that MMP is a transition state analog for the IMPDH reaction.