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Biomedical subjects

J S Nishimura

Publications and source records attributed to J S Nishimura.

At least 37 records · Page 2Linked to original sources
PubMed1983

Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine.

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.

Adenosine↗
PubMed1983

Affinity labeling of succinyl-CoA synthetase from Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate.

The 2'3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase. Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol alpha beta or, extrapolated, 2 mol/mol inactive alpha 2 beta 2 molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate in equilibrium succinyl-CoA exchange reaction, in comparison to the CoA in equilibrium succinyl-CoA exchange reaction. Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.

Adenosine Diphosphate↗
PubMed1983

A study of the quenching of the intrinsic fluorescence of succinyl-CoA synthetase from Escherichia coli by acrylamide, iodide, and coenzyme A.

Escherichia coli succinyl-CoA synthetase (SCS) contains three tryptophan residues per mole of alpha beta dimer, and all of them are on the beta subunit. SCS shows an emission maximum at 335 nm which is shifted to 350 nm upon denaturation by urea or guanidine hydrochloride. Acrylamide is able to quench the tryptophan fluorescence in SCS by static and dynamic mechanisms. Substrates give protection against quenching by acrylamide. Binding of ATP to the alpha subunit which has no tryptophans gives as large an effect on the quenching by acrylamide as the binding of coenzyme A (CoA) to the beta subunit. Addition of CoA eliminates the curvature observed in Stern-Volmer plots for acrylamide quenching obtained by lifetime measurements. Potassium iodide does not quench the SCS fluorescence in the presence of CoA. These results suggest that there are heterogeneously emitting tryptophan residues in SCS that are located at the alpha beta subunit contact region close to the CoA binding site. Hence, the tryptophan residues can act as intrinsic reporters of events taking place at the active site of this enzyme. Further, the present results support models for SCS that put the active site at the alpha beta subunit contact region.

Acrylamide↗
PubMed1982

Coenzyme A thiosulfonate (coenzyme A disulfide-S,S-dioxide), an affinity analog of coenzyme A.

The structure of the CoA affinity analog-oxidized CoA disulfide (o-CoAS2) (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4939) has been deduced to be that of the thiosulfonate of CoA, i.e. coenzyme A disulfide-S,S-dioxide. This deduction is based on several considerations among which are: the cleavage of o-CoAS2 by dithiothreitol under anaerobic conditions to equimolar amounts of CoASH and CoASO2H; the alkali-catalyzed dismutation of 3 mol of o-CoAS2 to 4 mol of CoASO2H and 1 mol of CoA disulfide; and comparison of the 13C-NMR spectra of CoA disulfide and o-CoAS2. The results of studies with Clostridial phosphotransacetylase (EC 2.3.1.8) and pigeon muscle carnitine acetyltransferase (EC 2.3.1.7) were consistent with the action of o-CoAS2 as a CoA affinity analog on these enzymes. Inactivation was characterized by what appeared to be disulfide bonding between CoA and important sulfhydryl groups of the proteins.

Affinity Labels↗
PubMed1980

Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits.

Affinity Labels↗
PubMed1979

Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli.

Ethoxyformic anhydride was used to demonstrate the existence of a second important histidine in succinyl-CoA synthetase from Escherichia coli. Differential labeling of the enzyme by [3H]ethoxyformic anhydride gave a stoichiometry of one important histidine per alpha beta catalytic unit. Data are presented suggesting that this residue and an important thiol group on the beta subunit (Collier, G., and Nishimura, J.S. (1978) J. Biol. Chem. 253, 4938-4943) interact with each other during catalysis. A mechanism of action involving these 2 residues is proposed for one of the partial reactions catalyzed by succinyl-CoA synthetase.

Binding Sites↗
PubMed1979

Acetate kinase from Veillonella alcalescens. Regulation by succinate and substrates.

The kinetic properties of acetate kinase from Veillonella alcalescens were investigated. In the presence of high concentrations of nucleotide both forward and reverse reactions were observed. In the presence of succinate the degree of cooperativity between subunits of the homodimer decreased, i.e. the Hill coefficient, n, decreased from 2.5 to 1.4 for acetyl phosphate in the presence of succinate. At low substrate concentrations hyperbolic kinetic data were observed with succinate. We have proposed a modified version of the concerted symmetry model to describe the kinetics observed with this enzyme. The primary differentiating feature of the proposed model is the requirement for activator ligand binding for catalysis. In the absence of succinate, the substrate (acetate or acetyl phosphate) also functions as an activating ligand.

Acetate Kinase↗
PubMed1979

Acetate kinase from Veillonella alcalescens. Purification and physical properties.

Acetate kinase (ATP:phosphotransferase E.C.2.7.2.1) has been purified to a high state of purity from Veillonella alcalescens. The native enzyme had a molecular weight of 88,000, as determined by Sephadex G-150 gel filtration. The molecular weight of the monomeric enzyme, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42,000. The enzyme was determined to be a homodimer from the amino acid composition and the results of trypsin digestion and cyanogen bromide cleavage. Two moles of phosphate were incorporated into the dimer upon incubation of the enzyme with ATP and acetate. These results support the conclusion that each subunit of the dimeric enzyme consists of a single active catalytic center. Succinate enhanced the rate of ATP-ADP phosphoryl group exchange 20-fold and the binding of ATP 10-fold. These results are considered in light of data from previous reports (Pelroy, R. A., and Whiteley, H. R. (1971) J. Bacteriol. 105, 259-267; Bowman, C. M., Valdez, R. O., and Nishimura, J. S. (1976) J. Biol. Chem 251, 3117-3121).

Acetate Kinase↗
PubMed1978

Affinity labeling of succinyl-CoA synthetase from porcine heart and Escherichia coli with oxidized coenzyme A disulfide.

Incubation of oxidized coenzyme A disulfide (produced by oxidation of reduced CoA with 1 eq of sodium periodiate or of CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA synthetase from either porcine heart or Escherichia coli led to the formation of inactive enzyme containing 1 mol of CoA per alphabeta dimer. The bound CoA was attached through a disulfide bond to a sulfhydryl group of the beta subunit. Release of CoA and restoration of activity was achieved by incubation of the modified enzyme with thiols, such as dithiothreitol. Interaction of oxidized CoA disulfide with enzyme was inhibited competitively by desulfo-CoA, which is a competitive inhibitor of the enzyme with respect to CoA. These data are evidence that oxidized CoA disulfide is an affinity label for the CoA binding site of succinyl-CoA synthetase and are the first positive results implicating the beta subunit in the catalytic mechanism of the enzyme.

Affinity Labels↗
PubMed1976

Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Acetate kinase of Veillonella alcalescens has been shown to be highly regulated enzyme exhibiting two levels of control: the requirement for succinate as a heterotropic allosteric effector, and cooperative binding at the substrate level. Succinate addition was necessary for enzymatic activity in both the direction of acyl phosphate synthesis and that of ATP synthesis. Control at the substrate level was apparent in the cooperative binding (Hill coefficients of 2) of acetyl phosphate, ATP, and ADP. Typical Michaelis kinetic data were observed for succinate (Ka = 20 mM for acetyl phosphate synthesis, 0.4 mM for ATP synthesis), acetate, and propionate. The primary effect of succinate was to increase the apparent Vmax of the enzymatic reaction for the variable substrates, ATP, ADP, and acetyl phosphate. The results are interpreted as evidence that, as a heterotropic effector of the acetate kinase reaction, succinate may regulate levels of propionyl-CoA (produced from propionyl phosphate by action of phosphotransacetylase), a compound required for the conversion of succinate to propionate. Acetase kinase has been shown to be a probable dimeric protein composed of two subunits of molecular weight 44,000 each.

Acetates↗
PubMed1975

Escherichia coli succinic thiolinase. Stoichiometry of phosphorylation and coenzyme A binding.

Equilibrium and covalent binding studies of succinic thiokinase from Escherichia coli indicates that there can be a stoichiometric relationship between coenzyme A binding and the phosphoyrlation capacity of the enzyme. A comparison of homogeneous enzyme preparations has revealed that enzyme of high specific activity exhibits greater binding capacity and that this property is proportional to enzyme activity. Phosphorylation capacity was related to specific activity in highly active enzyme preparations, but leveled off at 1 mol of phosphorus/mol of thiokinase. These studies show that the "dimer of dimers" structure of succinic thiokinase contains the expected two active sites and that this enzyme does not demonstrate "half-the-sites" reactivity. A coenzyme A binding site of lower affinity can be detected in some enzyme samples of lower specific activity. Binding of coenzyme A to the higher affinity sites may involve positive cooperativity. ATP, unlike ADP, does not bind to phosphorylated enzyme.

Adenosine Diphosphate↗
PubMed1975

Cross-linking of Escherichia coli succinic thiokinase. I. Reaction with diiminoesters and dimaleimides.

Treatment of the tetrameric alpha2 beta2 protein succinic thiokinase of Escherichia coli with dimethylsuberimidate (DMS) yielded five protein species detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These five protein species had estimated molecular weight values of 29,500, 41,000, 73,000, 117,000, and 132,000, and corresponded to alpha monomer, beta monomer, alpha beta dimer, alpha2 beta trimer and alpha2 beta2 tetramer, respectively. In all cases, the cross-linking produced predominantly the 73,000 molecular weight dimer with respectively lesser amounts of the tetramer and trimer. Succinic thiokinase was also cross-linked by reaction with N, N'-o-phynylenedimaleimide or with N, N'-P-phenylenedimaleimide. In these instances, treatment produced the alpha beta dimer as the only oligomeric species. Ammonolysis of isolated tetramer, trimer, and dimer produced by DMS treatment gave the 29,500 molecular weight monomer (alpha monomer) and the 38,500 molecular weight monomer (beta monomer). The absence of dimers of like subunits and the predominance of the dimer of unlike subunits are consistent with a quaternary structure of the native enzyme in which unlike subunits are closely associated but like subunits are not. Under certain conditions, an additional dimer of approximately 60,000 molecular weight was produced. This appeared to result from cleavage of the beta chain of an alpha beta dimer. Phosphorylation of native succinic thiokinase with [gamma-32P]ATP and [gamma-32P]GTP showed radioactivity only in the alpha monomer. Phosphorylation of enzyme before or after cross-linking showed radioactivity in all cross-linked bands except the beta monomer. Experiments in which the enzyme was titrated with [14C]DMS and trinitrobenzenesulfonate revealed that approximately half of the available amino groups reacted with the diiminoester, but that a small fraction of these (smaller than 20%) had reacted bifunctionally

Adenosine Triphosphate↗