Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.     

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.     

Regulation of citrate synthase from blue-green bacteria by succinyl coenzyme A

Citrate synthase (EC 4.1.3.7) was prepared from nine species of blue-green bacteria. In every case the citrate synthase was of the large type otherwise found only in Gram-negative bacteria.In addition to inhibition by -oxoglutarate, the enzymes were all sensitive to inhibition by succinyl coenzyme A, acting competitively with respect to acetyl coenzyme A. Desensitization by potassium chloride and a sigmoidal dependence of inhibition on succinyl coenzyme A concentration suggested the possibility of an allosteric mechanism. Multiple-inhibition analysis using pairs of the competitive inhibitors succinyl coenzyme A, bromoacetyl coenzyme A and ATP confirmed the existence of a distinct site for succinyl coenzyme A.It is suggested that the specific sensitivity of bluegreen bacterial citrate synthases to succinyl coenzyme A, as well as to -oxoglutarate, is related to the particular metabolic role of the enzyme in these organisms. The absence of a complete energy-yielding citric acid cycle, resulting from the lack of -oxoglutarate dehydrogenase, confers a strictly biosynthetic role on citrate synthase, which initiates a branched pathway leading to the two end-products -oxoglutarate and succinyl coenzyme A. Inhibition of the enzyme by these compounds constitutes a plausible regulatory mechanism.     

Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase.

(a) The reactivity of substituted acetates as substrates for CoA transferase increases sharply with increasing basicity and exhibits a slope of 1.0 in a plot of either log kappacat or log (kappacat/Km) against pKa (betanuc = 1.0). This result shows that the catalyzed reaction, which involves both carboxylate activation and leaving group transfer, does not proceed through a fully concerted reaction mechanism in the rate-determining step. The result is consistent with a stepwise reaction mechanism that proceeds through an anhydride intermediate. (b) Equilibrium constants for thiol ester formation, either bound to the enzyme or free in solution, show the same dependence on the basicity of carboxylate ions (betaeq = 1.0) and are independent of acidity when expressed in terms of the carboxylic acid. Thus, the polar environment around substituents on the acyl group is the same for carboxylic acids, thiol esters, and oxygen esters. (c) The interaction of the terminal CH3CO group of acetoacetate with the active site causes a 200,000-fold increase in kappacat/Km, corresponding to a decrease in delta G++ OF 7.2 kcal/mol compared with an unsubstituted acid of the same pK. The binding energy of the coenzyme A moiety of the substrate is utilized to interact with the active site and cause a 10(4) to 10(6)-fold increase in kappacat, corresponding to a decrease in delta G++ of 6 to 9 kcal/mol, compared with fragments of the coenzyme A moiety added separatly or together. (d) The exchange of labeled coenzyme A into acyl-CoA substrates was found to be greater than or equal to 10(5) slower than substrate turnover.     

Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.

A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.     

Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.

A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.     

Two functional domains of coenzyme A activate catalysis by coenzyme A transferase. Pantetheine and adenosine 3'-phosphate 5'-diphosphate

Studies of the reactivity of succinyl-CoA:3-keto acid CoA transferase with a small coenzyme A analog, methylmercaptopropionate, have shown that noncovalent interactions between the enzyme and the side chain of CoA are responsible for a rate acceleration of approximately 10(12), which is close to the total rate acceleration brought about by the enzyme (Moore, S. A., and Jencks, W. P. (1982) J. Biol. Chem. 257, 10893-10907). We report here that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of the rate acceleration and destabilization of the enzyme-thiol ester intermediate that is observed with CoA substrates. The role of the adenosine 3'-phosphate 5'-diphosphate moiety of CoA is to provide 6.9 kcal/mol of binding energy in order to pull the pantetheine moiety into the active site. The enzyme-thiol ester intermediate, E-pantetheine, was generated by reaction of pantetheine with the thiol ester of enzyme and methylmercaptopropionate. E-Pantetheine undergoes hydrolysis with khyd = 2 min-1, 140-fold faster than E-CoA, and reacts with acetoacetate with kAcAc = 3 X 10(6) M-1 min-1, only 10-fold slower than E-CoA. However, in the reverse direction acetoacetylpantetheine reacts with CoA transferase (kAcAc-SP = 220 M-1 min-1) 1.6 X 10(6) times slower than acetoacetyl-CoA. The equilibrium constant for the reaction of pantetheine with E-CoA is approximately 8 X 10(-6).     

Steady state kinetic mechanism of the Escherichia coli coenzyme A transferase.

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins when incubated in the dark in the presence of protoporphyrin and cofactors. These results constituted the first unambiguous demonstration of the insertion of magnesium into exogenous protoporphyrin in a cell-free system from higher plants. The metalloporphyrin synthetic activity did not occur in the absence of the plastids or when the plastids were heated in a 100 °C water bath for 2 min. It is thus suggested that, in higher plants, the in vitro insertion of magnesium into protoporphyrin is an enzymatic reaction.