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At least 199 records · Page 11Linked to original sources
PubMed1980

Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation.

The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by [3-14C]pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques. The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes. At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)). The slower process is much too slow to be of catalytic significance. The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed. The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase). The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation. Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur. The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway.

Acetylation↗
PubMed1980

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.

Escherichia coli↗
PubMed1980

Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides.

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

Amino Acids↗
PubMed1981

Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.

Amidohydrolases↗
PubMed1983

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Bacillus subtilis↗
PubMed1985

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis.

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.

Bacillus subtilis↗
PubMed1969

Crystallization of a multienzyme complex: fatty acid synthetase from yeast.

The crystallization of purified fatty acid synthetase from yeast is facilitated by seeding techniques. The first seed crystals appeared as particles with almost negligible enzymatic activity in a solution that had been left at 4 degrees C for 15 months. Subsequently, the crystallization time has been reduced to two days, and the crystals now isolated have retained their full enzymatic activity. The loss of synthetase activity that can result from prolonged exposure during crystallization is attributable specifically to damage of the enzyme component responsible for condensation activity. Crystalline synthetase is identical to purified but noncrystalline synthetase by all criteria so far examined.

Crystallization↗