Modulation of rat-liver mitochondrial acetyl-CoA acetyltransferase activity by a reversible chemical modification with coenzyme A

The mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) is involved in ketone body biosynthesis. In its unmodified state, referred to as transferase B in former publications (Huth, W. (1981) Eur. J. Biochem. 120, 557-562), the enzyme is characterized by the highest specific activity of 21.65 mumol/min per mg protein (direction of acetoacetyl-CoA synthesis); several forms of the enzyme with lower specific activities result from chemical modification by an apparent covalent binding of CoASH. The chemical modification results in an inactivation of the enzyme: a 2 h incubation with 0.2 mM CoASH at pH 8.1 at 30 degrees C inactivates up to 95%. Both processes, the CoASH-binding and the resulting inactivation, can be simultaneously reversed by treatment with glutathione. The specificity of inactivation is limited to CoASH and the intact sulfhydryl group is a prerequisite for this process. The enzyme exhibits a limited number (n = 3.2) of high-affinity (Ka = 26.7 microM) specific binding sites for CoASH. The inactivation-reactivation cycle of acetyl-CoA acetyltransferase by CoASH and glutathione may involve a protein disulfide-thiol exchange and represents a mode of control in modulating the amount of active enzyme.     

Mitochondrial acetyl-CoA acetyltransferase in liver and extrahepatic tissues: role of modification by coenzyme A

The influence of clofibrate and di(2-ethylhexyl)phthalate on mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA: acetyl-CoA C-acetyltransferase, EC 2.3.1.9), the rate-limiting ketogenic enzyme, which can be modified and inactivated by CoA, was investigated. In fed rats, both compounds induced a doubling of ketone bodies in the blood and, moreover, an increase by about 13% in the hepatic relative amount of the unmodified, i.e., the most active form of the enzyme (immunoreactive protein). This shift would account for an elevation of overall enzyme activity by about 5% only. Thus, the CoA modification of mitochondrial acetyl-CoA acetyltransferase did not explain the entire augmentation of ketone bodies. However, clofibrate and di(2-ethylhexyl)phthalate also increased the immunospecific protein and enzyme activity by approx. 2- and 3-fold, respectively. These effects were observed in liver, but not in several extrahepatic tissues.     

Densitization of pyruvate carboxylase against acetyl-CoA stimulation by chemical modification

Sheep kidney pyruvate carboxylase has been desensitized against its allosteric effector, acetyl CoA, by limited covalent modification with trinitrobenzene sulphonic acid.Trinitrophenylation of the enzyme resulted in a strong inhibition of the rate of the acetyl CoA-stimulated pyruvate carboxylation and enhancement of the rate of the acetyl CoA-independent reaction. A good correlation was found between the requirement for acetyl CoA of the exchange reactions catalysed by the enzyme and the extent of their inhibition by trinitrobenzene sulphonic acid modification.Spectrophotometric data indicated that one to two lysyl residues per monomer were trinitrophenylated. Modification had only a slight effect on the sedimentation properties of the enzyme.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification

The mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), which is involved in the biosynthesis or degradation of ketone bodies, was directly demonstrated in organ extracts applying a two-step chromatography-immunoelectrophoresis method. In liver, the enzyme can be shown in at least three forms: in an unmodified state, designated as AAT, and in the CoASH-modified forms A1 and A2, in amounts of 51.5 +/- 5.0%, 39.4 +/- 4.8% and 9.1 +/- 2.7% (areas of immunoprecipitation), respectively. This pattern, which could not be altered by a treatment with glutathione, resembles that of mitochondrial acetyl-CoA acetyltransferase in extrahepatic tissues. However, the proportion of the unmodified enzyme (AAT) is lower as compared to those in other tissues such as brain (81.5 +/- 4.4%). CoASH-modification and transformation into modified forms, which equal naturally occurring forms, can be demonstrated in vitro with acetyl-CoA acetyltransferase from both liver and brain. Thus CoASH-modification of mitochondrial acetyl-CoA acetyltransferase seems to be a process of general importance.     

Control of acetyl-CoA carboxylase by covalent modification

Summary In this review, various experiments which establish the occurrence of covalent modification mechanisms, both in vivo and in vitro, in the control of acetyl-CoA carboxylase have been presented. It is interesting to note that phosphorylation of the carboxylase results in disaggregation of the active species. These studies indicate that aggregation and disaggregation of the enzyme are involved in the control of carboxylase activity. Our covalent modification mechanism and the allosteric control mechanism share a common ground in that both mechanisms affect the equilibrium between protomers and polymers of the enzyme. However, it is clear that the allosteric control mechanism cannot functon alone under normal physiological conditions. Covalent modification of the carboxylase is prerequiste for efficient functioning of the allosteric mechanism.There are many aspects of the regulation of acetyl-CoA carboxylase which require further clarification. However, it is now established that short-term control of acetyl-CoA carboxylase involves the covalent modification mechanism.This research was supported by a grant from National Institutes of Health (AM 12865).This is Journal Paper No. 7701 from Purdue Agriculture Experiment Station.     

Acetyl coenzyme a-glutamate acetyltransferase and N-acetylornithine-glutamate acetyltransferase of chlorella

The enzymic formation of acetylglutamate has been studied in Chlorella vulgaris extracts. Acetyl CoA and N(2)-acetyl-l-ornithine served as substrates for glutamate acetylation whereas acetylphosphate, N(5)-acetyl-l-ornithine, and N(2)-acetyl-2,4-diamino butyrate were ineffective. Acetyl CoA-glutamate transacetylase and acetylornithine-glutamate transacetylase activities have been purified over 180-fold with no indication of any separation of activities. The acetyl CoA activity was more labile than acetylornithine activity so that preparations having acetylornithine-glutamate transacetylase activity but no acetyl CoA-glutamate transacetylase activity were obtained. The two acetylating activities appear to be properties of one enzyme with one portion more easily denatured.Both acetylating activities had pH optima between 8 and 8.5. The Km value for glutamate was 3 mm for both activities. The Km values were 0.2 mm for acetylornithine and 3.2 mm for acetyl CoA. Arginine inhibited acetyl CoA-glutamate transacetylase (Ki = 0.94 mm) and acetylglutamate phosphokinase (Ki = 0.5 mm) but had no effect on acetylornithine-glutamate transacetylase. The lack of an inhibitory effect of proline on any of the three enzymic activities indicates that acetylglutamate is not a normal intermediate in proline biosynthesis. Growth of Chlorella with arginine as a nitrogen source had no effect on enzyme levels, showing that end-product repression is not a control factor in arginine biosynthesis in Chlorella. In Chlorella, arginine controls its own biosynthesis by inhibiting acetylglutamate phosphokinase and controls the level of acetylated intermediates by inhibiting acetyl CoA-glutamate transacetylase.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Inhibition of lyso-PAF: acetyl-CoA acetyltransferase by salicylates and other compounds

Diflunisal and benoxaprofen (20-100 microM) produced dose-dependent inhibitions of lyso-platelet activating factor: acetyl-CoA acetyltransferase in a lysate of rat pleural neutrophils. Salicylate and aspirin were inhibitory at concentrations of 1 mM and above. Nordihydroguaiaretic acid was a relatively potent inhibitor (I50 = 6 microM). Other compounds, including anti-inflammatory steroids, cyclooxygenase and 5-lipoxygenase inhibitors, appeared ineffective at relevant concentrations. Inhibitions by diflunisal and salicylate occurred at concentrations similar to expected plasma levels in humans at therapeutic doses. An inhibition of platelet-activating factor synthesis may contribute to the antiinflammatory, analgesic, or antipyretic actions of these compounds.     

Characterization of acetyl-CoA: lyso-PAF acetyltransferase of human mesangial cells

Platelet activating factor (PAF) is a potent inflammatory mediator produced by various renal cells and it is implicated in renal pathology. The aim of this study is the characterization of remodeling lyso-PAF acetyltransferase, which is activated under inflammatory conditions, in human mesangial cell. Total membranes of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by trichloroacetic acid precipitation method. The effect of BSA, divalent cations, EDTA, and various chemicals on the activity of lyso-PAF acetyltransferase was also studied. Various detergents were also tested for the solubilization of the enzyme and only glycerol did not affect its activity. Partial purification of solubilized enzyme preparations of human kidney tissue and mesangial cells was performed on anion exchange column chromatography and native-PAGE electrophoresis and two active fractions were detected.     

Altering the interfacial activation mechanism of a lipase by solid-phase selective chemical modification

This study presents a combined protein immobilization, directed mutagenesis, and site-selective chemical modification approach, which was used to create a hyperactivated semisynthetic variant of BTL2. Various alkane chains were tethered at three different positions in order to mimic the lipase interfacial activation exogenously triggered by detergents. Optimum results were obtained when a dodecane chain was introduced at position 320 by solid-phase site-selective chemical modification. The resulting semisynthetic variant showed a 2.5-fold higher activity than the wild-type nonmodified variant in aqueous conditions. Remarkably, this is the maximum hyperactivation ever observed for BTL2 in the presence of detergents such as Triton X-100. We present evidence to suggest that the endogenous dodecane chain hyperactivates the enzyme in a similar fashion as an exogenous detergent molecule. In this way, we also observe a faster irreversible enzyme inhibition and an altered detergent sensitivity profile promoted by the site-selective chemical modification. These findings are also supported by fluorescence studies, which reveal that the structural conformation changes of the semisynthetic variant are different to those of the wild type, an effect that is more pronounced in the presence of detergent. Finally, the optimal immobilized semisynthetic variant was successfully applied to the selective synthesis of oxiran-2-yl butyrate. Significantly, this biocatalyst is 12-fold more efficient than the immobilized wild-type enzyme, producing the S-enantiomer with higher enantiospecificity (ee = 92%).     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase. Hydrophobic determinants of recognition and catalysis.

The preponderance of nonpolar contacts between CoA and chloramphenicol acetyltransferase in the high resolution structure of the binary complex prompted a study of selected hydrophobic residues by site-directed mutagenesis and steady-state kinetic analysis. Substitutions of three aromatic residues were used to evaluate binding contacts with the adenine moiety of CoA (Tyr-178), the pantetheine arm of the coenzyme (Tyr-56), and the S-acyl substituent (Phe-33). For those substitutions at residues 56 and 178 that cannot promote alternative polar interactions there is a correlation between residue hydrophobicity and the free energy of formation of the binary and ternary complexes of acetyl-CoA and chloramphenicol acetyltransferase and of the transition-state complex. Substitutions at Tyr-178 destabilize all such complexes to approximately the same extent (uniform binding changes), whereas those at Tyr-56 and Phe-33 cause differential binding changes, having a greater effect on the transition state than on either of the other complexes with acetyl-CoA.     

The mitochondrial tricarboxylate carrier of silver eel: chemical modification by sulfhydryl reagents

The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyl)maleimide (PM). Citrate and L-malate, two substrates of the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent PM bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.     

Nonhomogeneous labeling of liver mitochondrial acetyl-CoA

The specific activity of carbons 1 and 2 of plasma acetoacetate has been used as a measure of the specific activity of liver mitochondrial acetyl-CoA in tracer studies. To test whether or not acetoacetate actually reflects acetyl-CoA, livers were perfused with a mixture of substrates that are converted to mitochondrial acetyl-CoA: 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and, where indicated, 0.2 mM octanoate or 0.2 mM alpha-ketoisocaproate. In each experiment, one of these substrates was 13C-labeled. Labeling of mitochondrial acetyl-CoA was assessed by three methods: (i) molar percent enrichment of total tissue acetyl-CoA; (ii) molar percent enrichment of carbons 4 and 5 of tissue citrate, the precursor of which is acetyl-CoA; and (iii) molar percent enrichment of carbons 1 and 2 of perfusate ketone bodies. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA occurred under most conditions, i.e. the enrichments of carbons 4 and 5 of citrate were different from enrichments of carbons 1 and 2 of ketone bodies. Thus, based upon our results obtained in perfused livers, we question the validity of measuring the labeling of carbons 1 and 2 of acetoacetate as a noninvasive probe of liver mitochondrial acetyl-CoA.     

On the regulation of cold-labile cytosolic and of mitochondrial acetyl-CoA hydrolase in rat liver

The discovery of a cold-labile cytosolic acetyl-CoA hydrolase of high activity in rat liver by Prass et al. [(1980) J. Biol. Chem. 255, 5215-5223] has questioned the importance of mitochondrial acetyl-CoA hydrolase for the formation of free acetate [Grigat et al. (1979) Biochem. J. 177, 71-79] under physiological conditions. Therefore this problem has been reevaluated by comparing various properties of the two enzymes. Cold-labile cytosolic acetyl-CoA hydrolase bands with an apparent Mr of 68000 during SDS/polyacrylamide gel electrophoresis, while the native enzyme elutes in two peaks with apparent Mr of 136000 and 245000 during gel chromatography in the presence of 2 mM ATP. The mitochondrial enzyme elutes under the same conditions with an apparent Mr of 157000. Under conditions where the cold-labile enzyme binds strongly to DEAE-Bio-Gel and ATP-agarose, the mitochondrial enzyme remains unbound. The cold-labile enzyme can be activated 14-fold by ATP, half-maximal activation occurring already at 40 microM ATP. AdoPP[NH]P, AdoPP[CH2]P and GTP have a similar though weaker effect. ADP as well as GDP can completely inhibit the cold-labile enzyme with 50% inhibition occurring for both nucleotides at about 1.45 microM. The binding of ATP and ADP is competitive. Acetyl phosphate and pyrophosphate have no effect on the activity of the cold-labile enzyme. The mitochondrial acetyl-CoA hydrolase is not affected by these nucleotides. CoASH is a strong product inhibitor (approximately equal to 80% inhibition at 40 microM CoASH) of the cold-labile enzyme, but only a weak inhibitor of the mitochondrial enzyme. Under in vivo conditions the activity of the cold-labile cytosolic acetyl-CoA hydrolase can be no more than 7% of the activity calculated for mitochondrial acetyl-CoA hydrolase under the same conditions. Accordingly the mitochondrial enzyme seems to be mainly responsible for the formation of free acetate by the intact liver, especially in view of the fact that the substrate specificity of the mitochondrial enzyme is much higher (activity ratios acetyl-CoA/butyryl-CoA 4.99 and 1.16 for the mitochondrial and the cold-labile enzyme respectively). Alloxan diabetes neither increased the activity of the cold-labile enzyme nor that of the mitochondrial enzyme. No experimental support has been found yet for the hypothesis that the acetyl-CoA hydrolase activity of the cold-labile enzyme represents the side-activity of an acetyl-transferase.     

The synthesis of acetyl-CoA by Clostridium thermoaceticum from carbon dioxide,hydrogen, coenzyme A and methyltetrahydrofolate

It has been demonstrated that enzymes from Clostridium thermoaceticum catalyze the following reaction in which Fd is ferredoxin and CH3THF is methyltetrahydrofolate. (for formula see text). The system involves hydrogenase, CO dehydrogenase, a methyltransferase, a corrinoid enzyme and other unknown components. Hydrogenase catalyzes the reduction of ferredoxin by H2; CO dehydrogenase then uses the reduced ferredoxin to reduce CO2 to a one-carbon intermediate that combines with CoASH and with a methyl group originating from CH3THF to form acetyl-CoA. It is proposed that these reactions are part of the mechanism which enables certain acetogenic autotrophic bacteria to grow on CO2 and H2.