Evidence for an arginine residue at the coenzyme-binding site of <i>Escherichia coli</i> isocitrate dehydrogenase

McKEE, J. S. McKINLEY; Nimmo, Hugh G.

doi:10.1042/bj2610301

Cited by 10 publications

(4 citation statements)

References 20 publications

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“…The phenylglyoxal-arginine product formed in Ap4A phosphorylase I is unstable under the conditions required for amino acid analysis and isolation of phenylglyoxal-labelled peptides. Similar instability of phenylglyoxal-arginine products in other enzymes has been reported (Jornvall et al, 1977;McKee & Nimmo, 1989). However, the stability of the inactivated enzyme to high dilution, precipitation with trichloroacetic acid and dissociation in SDS support the kinetic data that phenylglyoxal acts as an irreversible inhibitor of Ap4A phosphorylase I.…”

Section: Discussionsupporting

confidence: 83%

Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae

Robinson

Barnes

1991

Biochemical Journal

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Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner. The second-order rate constant was 11.5 +/- 2.5 M-1 min-1. The loss of activity was a linear function of the incorporation of [7-14C]phenylglyoxal. The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity. The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi. The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not. Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively. The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal. Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A.

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Section: Discussionsupporting

confidence: 83%

Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae

Robinson

Barnes

1991

Biochemical Journal

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“…Earlier studies with model compounds found that the stoichiometry of the reaction of phenylglyoxal with arginine requires 2 mol of phenylglyoxal for each molar equivalent of arginine residue (Takahashi, 1968;Lange et al, 1974;Cheng & Nowak, 1989). However, examples are also known where a 1:1 stoichiometry was found, depending on the particular local conformation of the enzyme active site (Borders & Riordan, 1975;Gildensoph & Briskin, 1989;McKee & Nimmo, 1989). In any event, it appears that at least one arginine is essential for BHPTP activity.…”

Section: Resultsmentioning

confidence: 99%

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase

Zhang¹,

Davis

Etten

1992

Biochemistry

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Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.

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“…9). The activity of ICD also regulates the flux split between the full TCA cycle and the glyoxylate shunt (49)(50)(51). A point mutation at residue 395 that changed the amino acid from positively charged (L-arginine) to negatively charged (L-cysteine) in ICD was found in all ePgi replicates except replicate 7 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiple Optimal Phenotypes Overcome Redox and Glycolytic Intermediate Metabolite Imbalances in Escherichia coli pgi Knockout Evolutions

McCloskey

Sandberg

et al. 2018

Appl Environ Microbiol

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A mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory functions of the lost gene. The gene, whose product catalyzes the second step in glycolysis, was deleted in a growth-optimized K-12 MG1655 strain. The initial knockout (KO) strain exhibited an 80% drop in growth rate that was largely recovered in eight replicate, but phenotypically distinct, cultures after undergoing adaptive laboratory evolution (ALE). Multi-omic data sets showed that the loss of substantially shifted pathway usage, leading to a redox and sugar phosphate stress response. These stress responses were overcome by unique combinations of innovative mutations selected for by ALE. Thus, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed. A mechanistic understanding of how microbes are able to overcome the loss of a gene through regulatory and metabolic changes is not well understood. Eight independent adaptive laboratory evolution (ALE) experiments with knockout strains resulted in eight phenotypically distinct endpoints that were able to overcome the gene loss. Utilizing multi-omics analysis, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed.

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Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase

Cited by 10 publications

References 20 publications

Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae

Chemical modification of a functional arginine residue in diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase

Multiple Optimal Phenotypes Overcome Redox and Glycolytic Intermediate Metabolite Imbalances in Escherichia coli pgi Knockout Evolutions

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