Alkylation of an active-site cysteinyl residue during substrate-dependent inactivation of Escherichia coli S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.     

Effect of the hinge protein on the heme iron site of cytochrome c1

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].     

Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.     

Reassessment of 14CO2 compartmentation and of [14C]formate oxidation in rat liver

Our previous report (Marsolais, C., Huot, S., David, F., Garneau, M., and Brunengraber, H. (1987) J. Biol. Chem. 262, 2604-2607) had concluded that a fraction of [14C]formate oxidation in liver occurs in the mitochondrion. This conclusion was based on the labeling patterns of urea and acetoacetate labeled via 14CO2 generated from [14C]formate and other [14C]substrates. We reassessed our interpretation in experiments conducted in (i) perifused mitochondria and (ii) isolated livers perfused with buffer containing [14C]formate, [14C]gluconolactone, 14CO2, or NaH13CO3, in the absence and presence of acetazolamide, an inhibitor of carbonic anhydrase. Our data show that the cytosolic pools of bicarbonate and CO2 are not in isotopic equilibrium when 14CO2 is generated in the cytosol or is supplied as NaH14CO3. We retract our earlier suggestion of a mitochondrial site of [14C]formate oxidation.     

Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli.

The acetoacetyl-CoA-thiolase, a product of the acetoacetate degradation operon (ato) was purified to homogeneity as judged by polyacrylamide-gel electrophoresis at pH 4.5, 7.0, and 8.3. The enzyme has a molecular weight of 166,000 and is composed of four identical subunits. The subunit molecular weight is 41,500. Histidine was the sole N-terminal amino acid detected by dansylation. The thiolase contains eight free sulhydryl residues and four intrachain disulfide bonds per mole. The ato thiolase catalyzes the CoA- dependent cleavage of acetoacetyl-CoA and the acetylation of acetyl-CoA to form acetoacetyl-CoA. The maximal velocity in the direction of acetoacetyl-CoA cleavage was 840 nmol min^? (enzyme unit)^?1 and the maximal velocity in the direction of acetoacetyl CoA formation was 38 nmol min^?1 (enzyme unit)^?1. Like other thiolases, the ato thiolase was inactivated by sulfhydryl reagents. The enzyme was protected from inactivation by sulfhydryl reagents in the presence of the acyl-CoA substrates, acetyl-CoA and acetoacetyl-CoA; however, no protection was obtained when the enzyme was incubated with the acetyl-CoA analog, acetylaminodesthio-CoA. Consistent with these results was the demonstration of an acetyl-enzyme compound when the thiolase was incubated with [1-¹⁴C]acetyl-CoA. The sensitivity of the acetyl-enzyme bond to borohydride reduction and the protection afforded by acyl-CoA substrates against enzyme inactivation by sulfhydryl reagents indicated that acetyl groups are bound to the enzyme by a thiolester bond.     

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.     

Inactivation of pig heart NADP-specific isocitrate dehydrogenase by two affinity reagents is due to reaction with a cysteine not essential for function.

Pig heart NADP-dependent isocitrate dehydrogenase is 65% inactivated by 3-bromo-2-ketoglutarate (Ehrlich, R.S., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,614-12,619) and 90% inactivated by 2-(4-bromo-2,3-dioxobutylthio)-1,N6- ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) (Bailey, J.M., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,620-12,626). Both inactivation reactions result in enzyme with an incorporation of 1.0 mol reagent/mol enzyme dimer and both modified enzymes bind only 1.0 mol manganous isocitrate or NADPH/mol enzyme dimer as compared to 2.0 mol manganous isocitrate or NADPH/mol enzyme dimer for unmodified enzyme. The inactivation reactions, which occur at or near the nucleotide binding site, are mutually exclusive. Reaction with either affinity reagent led to the isolation of the same modified triskaidekapeptide, DLAGXIHGLSNVK. We have isolated from isocitrate dehydrogenase a peptide, DLAGCIHGLSNVK, that had been modified by N-ethylmaleimide (NEM) with no loss of enzymatic activity. We now show that enzyme modified by NEM in the presence of isocitrate plus Mn2+ retains full catalytic activity but is not inactivated by either of the affinity reagents; thus, all three reagents appear to react at the same site. The analysis of HPLC tryptic maps of isocitrate dehydrogenase treated under denaturing conditions with iodo[3H]acetic acid or [3H]NEM demonstrates that both bromoketoglutarate and 2-BDB-T epsilon A-2',5'-DP react with the cysteine residue of DLAGCIHGLSNVK. We conclude that the cysteine of this triskaidekapeptide is close to the coenzyme binding site but is not essential for catalytic function.     

The shunt pathway of mevalonate metabolism in the isolated perfused rat kidney

The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

Cloning and sequence determination of cDNA encoding a second rat liver peroxisomal 3-ketoacyl-CoA thiolase

3-Ketoacyl-coenzyme A thiolase (thiolase) catalyzes the final step of the fatty acid beta-oxidation pathway in peroxisomes. Thiolase is unique among rat liver peroxisomal enzymes in that it is synthesized as a precursor possessing a 26-amino acid (aa) N-terminal extension which is cleaved to generate the mature enzyme. To facilitate further examination of the synthesis, intracellular transport and processing of this enzyme, cDNA clones were selected from a lambda gt11 rat liver library using antiserum raised against peroxisomal thiolase. Upon sequencing several cDNA clones, it was revealed that there are at least two distinct thiolase enzymes localized to rat liver peroxisomes, one identical to the previously published rat liver peroxisomal thiolase (thiolase 1) [Hijikata et al., J. Biol. Chem. 262 (1987) 8151-8158] and a novel thiolase (thiolase 2). The THL2 cDNA possesses a single open reading frame of 1302 nucleotides (nt) encoding a protein of 434 aa (Mr 44790). The coding region of THL2 cDNA exhibits 94.6% nt sequence identity with THL1 and 95.4% identity at the level of aa sequence. Northern-blot analysis indicates that the mRNA encoding thiolase 2 is approx. 1.7 kb in size. The mRNA encoding thiolase 2 is induced approx. twofold upon treatment of rats with the peroxisome-proliferating drug, clofibrate. In contrast, the thiolase 1 mRNA is induced more than tenfold under similar conditions.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo.

Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a pI identical to the most basic adult alpha variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985-5991], which has sequence-motifs related to calponin, displays a similar isoform pattern during development; one isoform (alpha) is present in the embryo and three in the adult. In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32PO4, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65-68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10148-10155]. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (alpha) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the alpha isoform and a second more basic spot, not observed in tissue samples. Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the alpha SM 22 isoform during smooth-muscle phenotype modulation in vitro.     

Selective deacylation of arachidonate-containing ethanolamine-linked phosphoglycerides in stimulated human neutrophils

The involvement of the ethanolamine-linked phosphoglyceride fraction (PE) in neutrophil signal transduction is suggested by the stimulus-induced release of arachidonic acid from PE (Chilton, F. H., and Connell, T. R. (1988) J. Biol. Chem. 263, 5260-5265) and by the synthesis of acetylated PE species, predominantly 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alkenylacetyl-GPE; Tessner, T. G., and Wykle, R. L. (1987) J. Biol. Chem. 262, 12660-12664) in stimulated cells. In the studies reported here, we investigated the relationship between arachidonic acid release from PE and generation of the lysophospholipid precursor required in the biosynthesis of alkenylacetyl-GPE. In order to follow these reactions, we prelabeled neutrophils with 1-O-[3H]alk-1'-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (alkenyl-acyl-GPE). We also followed the hydrolysis of endogenous PE by analysis as the dinitrophenyl derivative using a high pressure liquid chromatography method we developed. Our results coupled with those of Chilton et al. (Chilton, F. H., Ellis, J. M., Olson, S. C., and Wykle, R. L. (1984) J. Biol. Chem. 259, 12014-12019) indicate that in human neutrophils the metabolism of alkenylacyl-GPE and alkylacyl-sn-glycero-3-phosphocholine (GPC) are strikingly similar with regard to arachidonate metabolism. When added to neutrophils, both 1-O-[3H]alkenyl-2-lyso-GPE and 1-O-[3H]alkyl-2-lyso-GPC are acylated predominantly with arachidonic acid, and the resulting arachidonoyl-containing phospholipids are extensively deacylated upon stimulation. However, hydrolysis of PE in the neutrophil differs from hydrolysis of choline-containing phosphoglycerides in that stimulation leads to a greater accumulation of the ethanolamine-linked lysophospholipid. A comparison of the molecular species of endogenous PE (based on molar concentrations measured as the dinitrophenyl derivative) from resting and stimulated neutrophils indicated that only those species which contain arachidonate are significantly hydrolyzed.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Identification and characterization of the dihydropyridine-binding subunit of the skeletal muscle dihydropyridine receptor

Photoaffinity labeling of isolated triads and purified dihydropyridine receptor with [3H]azidopine and (+)-[3H]PN200-110 has been used to identify and characterize the dihydropyridine-binding subunit of the 1,4-dihydropyridine receptor of rabbit skeletal muscle. The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads contains four protein subunits of 175,000, 170,000, 52,000, and 32,000 Da (Leung, A., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). Photoaffinity labeling of isolated triads with [3H]azidopine resulted in specific and covalent incorporation of [3H]azidopine into only the 170,000-Da subunit of the dihydropyridine receptor and not into the 175,000-Da glycoprotein subunit of the receptor. The [3H]azidopine-labeled 170,000-Da subunit was separated from the 175,000-Da glycoprotein subunit by sequential elution from a wheat germ agglutinin-Sepharose column with 1% sodium dodecyl sulfate followed by 200 mM N-acetylglucosamine. Photoaffinity labeling of purified dihydropyridine receptor with [3H]azidopine or (+)-[3H]PN200-110 also resulted in the specific and covalent incorporation of either ligand into only the 170,000-Da subunit. Therefore, our results show that the dihydropyridine-binding subunit of the skeletal muscle 1,4-dihydropyridine receptor is the 170,000-Da subunit and not the 175,000-Da glycoprotein subunit.     

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.     

Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.