title: Active-site modification of native and mutant forms of inosine 5'-monophosphate dehydrogenase from Escherichia coli K12.
abstract: IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-beta-d-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP>>AMP. NAD(+) did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in lambda(max.) of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A(290) with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2-3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[(14)C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[(14)C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors*; D007291:Inosine Monophosphate / Q000031:analogs & derivatives*; D007460:Iodoacetamide / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D011685:Purine Nucleotides / Q000494:pharmacology*; D013053:Spectrophotometry
chemical_list: D007527:Isoenzymes; D009711:Nucleotides; D011685:Purine Nucleotides; D007291:Inosine Monophosphate; C018488:6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate; D007168:IMP Dehydrogenase; D007658:Ketone Oxidoreductases; D007460:Iodoacetamide