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

Biochemistry. 1997 Oct 28;36(43):13365-73. doi: 10.1021/bi9714161.


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 kcat, Km, and Ki for GMP, XMP, mizoribine 5'-monophosphate (MMP), and beta-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 kcat and a 25-fold increase in the value of Kii 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 kcat, but only a 5-10-fold increase in the values of Km for IMP and Kis 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 Ki for MMP correlate with changes in kcat/KmKm 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence* / genetics
  • Animals
  • Aspartic Acid / genetics
  • Binding Sites / genetics
  • Carboxylic Acids / chemistry*
  • Conserved Sequence* / genetics
  • Cross-Linking Reagents
  • Enzyme Activation / drug effects
  • Enzyme Activation / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Ethyldimethylaminopropyl Carbodiimide
  • Glutamic Acid / genetics
  • Humans
  • IMP Dehydrogenase / antagonists & inhibitors
  • IMP Dehydrogenase / chemistry*
  • IMP Dehydrogenase / genetics
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Ribonucleosides / pharmacology


  • Carboxylic Acids
  • Cross-Linking Reagents
  • Ribonucleosides
  • Aspartic Acid
  • Glutamic Acid
  • mizoribine
  • IMP Dehydrogenase
  • Ethyldimethylaminopropyl Carbodiimide