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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

The citrate synthases of the gram-negative bacteria, Escherichia coli and Acinetobacter anitratum, are allosterically inhibited by NADH. The kinetic properties, however, suggest that the equilibrium between active (R) and inactive (T) conformational states is shifted toward the T state in the E. coli enzyme. We have now manipulated the cloned genes for the two bacterial enzymes to produce two chimeric proteins, in which one folding domain of each subunit is derived from each enzyme. One chimera (the large domain from A. anitratum and the small domain from the E. coli enzyme) is designated CS ACI::eco; the other is called CS ECO::aci. Both chimeras are roughly as active as the wild type parents, but their Km values for both substrates are lower than those for the E. coli enzyme, and NADH inhibition is markedly sigmoid, while that for E. coli citrate synthases is hyperbolic. Curve-fitting to the allosteric equation suggests that these differences are the result of the destabilization of the T state in the chimeras. The ACI::eco chimera exists almost entirely as a hexamer, like the A. anitratum enzyme, while the ECO::aci chimera, like the E. coli synthase, forms three major bands on nondenaturing polyacrylamide gels, two of them hexamers of different net charge, and one a dimer. These findings indicate that subunit interactions leading to hexamer formation in allosteric citrate synthases of gram-negative bacteria involve mainly the large domains. The chimeras are also used to show that the NADH binding site of E. coli citrate synthase is located entirely in the large domain. Sensitivity of the chimeras to denaturation by urea, to which the A. anitratum enzyme is much more resistant than the E. coli enzyme, is determined by the large domains. Sensitivity to inactivation by subtilisin is intermediate between those shown by the E. coli (very sensitive) and A. anitratum (quite resistant) synthases. This result suggests that digestibility by subtilisin is determined by conformational factors as well as the amino acid sequences of the target regions.
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PMID:Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes. 152 32

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.
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PMID:Topography of rat hepatic microsomal enzymatic components of the fatty acid chain elongation system. 254 Jan 64

Citrate synthase from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.
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PMID:Proteolysis of acinetobacter citrate synthase by subtilisin. 633 74

Limited proteolysis of citrate synthase by Astacus protease, chymotrypsin, clostripain, subtilisin and trypsin on primary fragmentation all yielded similarly sized large (Mr 35 000-36 000) and small fragments (Mr 13 500-14 000) but endoproteinase Lys-C gave fragments of Mr 40 500 and Mr 6500. The sites of the proteolytic attack were determined by Edman degradation of the fragmented synthase preparations, Chymotrypsin, subtilisin, trypsin and endoproteinase Lys-C hydrolyse the synthase at positions 323-324 (-Leu-Arg-), 321-322 (-Ala-Val-)/322-323 (-Val-Leu-), 313-314 (-Arg-Val-) and 366-367 (-Lys-Ala-), respectively. Chymotrypsin and subtilisin attack the small domain of the synthase at the loop between helices O and P very near to a catalytic residue, His-320, and abolish all synthase activities. Primary fragmentation by endoproteinase Lys-C and trypsin reduces the catalytic activity in the physiological overall reaction. Both fragmented enzyme species catalyse the hydrolysis and C-C bond cleavage reactions of citryl-CoA in a stimulated fashion compared to the steady-state rates of the native enzyme, and without hysteretic behaviour. The proteolytic cleavage occurs at acetyl-CoA binding sites within the small domain at the loops connecting helices O to P (trypsin) and Q to R (endoproteinase Lys-C) and reduces the affinity of acetyl-CoA. All of the altered kinetic properties of the fragmented enzyme species are related to this reduced affinity. The correlation between structure and function indicated above is strengthened by the unaltered affinity of oxaloacetate towards the fragmented synthase species. None of the proteolytic enzymes applied attacks oxaloacetate binding sites as defined by the structural work. Oxaloacetate inhibits the hydrolysis of citryl-CoA by the fragmented synthases (endoproteinase Lys-C, trypsin) competitively. An explanation is proposed. The isolated small and large fragments (endoproteinase Lys-C, trypsin) were enzymically inactive. Enzymic activity was restored on recombination of the fragments under denaturing conditions. Cleavage of the loops between helices O to P and Q to R by sequential fragmentation with endoproteinase Lys-C and trypsin inactivated the synthase completely. This result lends support to the idea that the open and closed crystal forms of the structural work are interconverted during the catalytic cycle.
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PMID:Hysteretic behaviour of citrate synthase. Site-directed limited proteolysis. 638 Oct 53

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.
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PMID:Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin. 677 58

The detailed proof of the 437-residue amino acid sequence (Mr 48,969) of porcine heart citrate synthase (EC 4.13.7) is described. The S-carboxymethylated protein has been cleaved at methionine (cyanogen bromide) and arginine (trypsin digest of citraconylated enzyme) residues to yield 14 and 17 major peptides, respectively. Peptides were initially fractionated by gel filtration, and those useful for sequence analysis were purified by high-performance liquid chromatography. Sequence analyses were performed on these primary peptides and on subpeptides generated by cleavage with the bromine adduct of 2-[(2-nitrophenyl)sulfenyl]-3-methylindole, Staphylococcus aureus V8 protease, trypsin, chymotrypsin, or acid. The overall sequence was confirmed by analyzing products of cleavage by hydroxylamine, acid, and subtilisin. A novel feature of the sequence is the identification of trimethyllysine at residue 368.
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PMID:Complete amino acid sequence of porcine heart citrate synthase. 709 27