title: 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 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.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Kerr
authors.forename: L
authors.initials: L
authors.lastname: Hedstrom
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002264:Carboxylic Acids / Q000737:chemistry*; D017124:Conserved Sequence / Q000235:genetics; D003432:Cross-Linking Reagents; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005022:Ethyldimethylaminopropyl Carbodiimide; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012263:Ribonucleosides / Q000494:pharmacology
chemical_list: D002264:Carboxylic Acids; D003432:Cross-Linking Reagents; D012263:Ribonucleosides; D001224:Aspartic Acid; D018698:Glutamic Acid; C010052:mizoribine; D007168:IMP Dehydrogenase; D005022:Ethyldimethylaminopropyl Carbodiimide