Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

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Abstract
IMP dehydrogenase of E. 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 .mchgt. 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 290 nm, 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 A290 with time was 1st-order. Thus it appears that Cl-IMP reacts with only 1 species of thiol at the IMP-binding site of the enzyme: 2-3 mol of Cl-IMP were bound/mol of IMP dehydrogenase tetramer. Of 10 mutant enzymes from guaB strains, 6 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 methanethiosulfonate 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 methanethiosulfonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[14C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[14C]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.

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