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.

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

“…IMPDH, encoded by the guaB gene catalyses the reversible conversion of IMP to XMP. In E. coli, at least two different enzymic pathways lead to the synthesis of XMP (Neuhard & Nygaard, 1996;Pimkin et al, 2009) which are well characterized (Gilbert et al, 1979;Gilbert & Drabble, 1980;Kerr & Hedstrom, 1997;Kerr et al, 2000;Tiedeman & Smith, 1985). However, the M. tuberculosis H37Rv purine de novo biosynthesis pathway enzymes have received little attention to date and are poorly studied.…”

IMP Dehydrogenase: Structure, Mechanism, and Inhibition

“…Reagents such as iodoacetamide and methylmethanethiosulphonate inactivate IMPDH. IMP protects against inactivation, which provided the first evidence that a Cys residue might be present in the active site 71,92. Surprisingly given the position of IMPDH at the junction of guanine nucleotide metabolism, no allosteric regulators have been identified for IMPDH (reports that ATP is an allosteric regulator have not been confirmed 5,63,79).…”

“…However, the catalytic Cys can also provide specificity. Several analogs of IMP have been designed to form covalent adducts with this residue, including 6-Cl-IMP, EICARMP, 2-Cl-methyl-IMP, 6-thio-IMP and 2-vinyl-IMP, 2-F-vinyl-IMP (Chart 11) 92,113,252-256. All of these compounds are time-dependent irreversible inactivators of IMPDH.…”

Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin

“…The degree of sensitivity exhibited by UCA synthase to these reagents is much lower than that of the IMP dehydrogenases. Both the human and E. coli IMP dehydrogenases, for example, are rapidly inactivated by iodoacetamide (1,6). In contrast, UCA synthase retains 87% of its activity after exposure to iodoacetamide for a period of 6 h. On the other hand, UCA synthase is completely inactivated by a 15-min exposure to p-HMB or mercuric chloride.…”

“…This result indicates that one or more thiol groups are necessary for the activity of the enzyme. The affinity reagent 6-chloro-IMP has been reported to rapidly inactivate the E. coli (6), human (1), and T. foetus IMP dehydrogenases (13). The inactivated E. coli enzyme could be quickly reactivated by incubation with 2-mercaptoethanol, and an active-site cysteine residue was implicated as the target of the 6-chloro-IMP.…”