Phosphorylation and formation of hybrid enzyme species test the "half of sites" reactivity of Escherichia coli succinyl-CoA synthetase

Recent sequencing experiments have identified alpha-His246 as the phosphorylation site of Escherichia coli succinyl-CoA synthetase [Buck, D., Spencer, M. E., & Guest, J. R. (1985) Biochemistry 24, 6245-6252]. We have replaced alpha-His246 with an asparagine residue using site-directed mutagenesis techniques. The resulting mutant enzyme (designated H246N) exhibited no enzyme activity, as expected, but was found as a structurally intact, stable tetramer. Small differences in the net charge of H246N and wild-type enzymes were first detected on native polyacrylamide gels. These charge differences were resolved by using native isoelectric focusing gels to further separate the wild-type enzyme into diphosphorylated, monophosphorylated, and unphosphorylated species. The enzyme species were found to be interconvertible upon incubation with the appropriate enzyme substrate(s). Sample mixtures containing increasing molar ratios of H246N (alpha H246N beta)2 to wild-type enzyme (alpha beta)2 were unfolded and then refolded. The refolded enzyme mixtures were analyzed for enzymatic activity and separated on native isoelectric focusing gels. The hybrid enzyme (alpha beta alpha H246N beta) retained a significant amount of enzyme activity and also exhibited substrate synergism (stimulation of succinate in equilibrium succinyl-CoA exchange in the presence of ATP). Substrate synergism with this enzyme has been interpreted as evidence for interaction between active sites in such a way that only a single phosphoryl group is covalently attached to the enzyme at a given time [Wolodko, W. T., Brownie, E.R., O'Connor, M. D., & Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119]. On the contrary, we conclude that tetrameric succinyl-CoA synthetase from E. coli is comprised of two independently active dimer molecules associated together to form a "dimer of dimers" that displays substrate synergism within each dimer and not necessarily between dimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further purification,characterization and salt activation of acyl-CoA synthetase fromEscherichia coli

Acyl-CoA synthetase was further purified fromEscherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B. The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200. Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000. The sedimentation coefficient was calculated as 4.4. S. An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum. The molar extinction coefficient was 9.2 · 10⁴. Kinetic constants were determined fortrans fatty acids. All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system. In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions. The inhibitory effect of chaotrope on the enzyme activity was reversible. These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).     

Further purification and characterization of human 3-methyladenine-DNA glycosylase. Evidence for broad specificity

Further purification of a human placental 3-methyladenine-DNA glycosylase by phosphocellulose column chromatography yielded a 6000-fold increase in specific activity with greater than 5% recovery. Although 3-methyladenine was the predominant base released from double-stranded methylated DNA by this enzyme, minor releasing activities for 7-methylguanine and 3-methylguanine were also observed. During purification, the three DNA glycosylase activities consistently copurified with constant ratios of specific activity. Moreover, all the activities were heat-inactivated at 50 degrees C at the same rate, required double-stranded methylated DNA as substrate, were inhibited by spermine and spermidine, and were not subject to product inhibition. These data strengthen the likelihood that the three activities are associated with a single DNA glycosylase.     

Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.     

The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.     

Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Carbamylphosphate synthetase from Salmonella typhimurium. Regulations, subunit composition, and function of the subunits.

Carbamylphosphate synthetase was purified to homogeneity from a derepressed strain of Salmonella typhimurium by a procedure based on affinity chromatography employing immobilized glutamine. The enzyme catalyzes the synthesis of carbamylphosphate from either ammonia or glutamine together with ATP and bicarbonate. The ATP saturation curve of either nitrogen donor is sigmoidal (n equals 1.5) but the affinity for ATP is higher with ammonia. In addition to the feedback inhibition by UMP and activation by ornithine which we previously reported (1), the activity was found to be stimulated by IMP and phosphoribosyl-1-pyrophosphate. Evidence from pool measurements in enteric bacteria by others suggests that of the latter two compounds only phosphoribosyl-1-pyrophosphate is physiologically significant. All effectors regulate enzyme activity by altering its affinity for ATP. Glutamine also modulates the affinity for ATP; it is increased as glutamine concentratiions decrease, an effect that could serve to insulate the cell against major changes in carbamylphosphate synthesis in response to fluctuations in concentration of glutamine. The molecular weight of the holoenzyme was estimated to be 150,000 by sucrose density gradient centrifugation in triethanolamine and Tris-acetate buffers in which the enzyme is a monomer. In the presence of ornithine in potassium phosphate buffer, the enzyme is an oligomer with a molecular weight of 580,000. This transition has been exploited as an alternate route of purifying the enzyme to homogeneity using successive sucrose density centrifugation. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate shows that the enzyme consists of two unequal subunits with molecular weights of 110,000 and 45,000. The two subunits were separated by gel filtration in the presence of 1 M potassium thiocyanate, ATP, MgCl2, glutamine, NH4Cl, ornithine, and UMP. The heavy subunit catalyzes the synthesis of carbamylphosphate from ammonia but not glutamine. The ATP saturation curve for the separated heavy subunit is still sigmoidal (n equals 1.4 and So.5 equals 0.3 mM). The ammonia dependent activity of the heavy subunit is stimulated by the activators ornithine, IMP, and phosphoribosyl-1-pyrophosphate but is only marginally inhibited by high concentrations of UMP. The addition of the light subunit restored full ability to utilize glutamine as well as normal sensitivity to UMP. Purified subunits were used for in vitro complementation studies with strains carrying mutations in pyrA, the structural gene encoding carbamylphosphate synthetase. The results indicate that the pyrA region encodes both subunits and that the structural genes for the two polypeptides are linked. A deletion mutant lacking both subunits of carbamylphosphate synthetase also lacked any ability to synthetize carbamylphosphate from ammonia. Hence, unlike certain other bacteria, S. typhimurium does not possess a carbamate kinase.     

Further purification and characterization of human 3-methyladenine-DNA glycosylase Evidence for broad specificity

Further purification of a human placental 3-methyladenine-DNA glycosylase by phosphocellulose column chromatography yielded a 6000-fold increase in specific activity with greater than 5% recovery. Although 3-methyladenine was the predominant base released from double-stranded methylated DNA by this enzyme, minor releasing activities for 7-methylguanine and 3-methylguanine were also observed. During purification, the three DNA glycosylase activities consistently copurified with constant ratios of specific activity. Moreover, all the activities were heat-inactivated at 50°C at the same rate, required double-stranded methylated DNA as substrate, were inhibited by spermine and spermidine, and were not subject to product inhibition. These data strengthen the likelihood that the three activities are associated with a single DNA glycosylase.     

Phenylalanyl-tRNA synthetase fromE. coli MRE-600: Localization of the phenylalanine binding sites on the subunits by affinity reagents

The phenylalanine and the phenylalanyl-tRNA^Phe binding sites on the subunits of phenylalanyl-tRNA synthetase fromE.coli MRE-600 were localized using p-azidoanilidate of [¹⁴C]phenylalanine and N-bromoacetyl[¹⁴C]phenylalanyl-tRNA^Phe. The phenylalanine recognizing site was shown to be situated on the subunit of the enzyme in close proximity to the contact region of the and subunits and the phenylalanyl-tRNA^Phe recognizing site on the subunit. Transfer of the aminoacyl moiety from the subunit to the subunit of the enzyme was assumed to take place in the process of catalysis of the aminoacylation reaction.     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

2'-Versus 3'-OH specificity in tRNA aminoacylation. Further support for the "secondary cognition" proposal.

Purified Escherichia coli tRNAAla and tRNALys were each converted to modified species terminating in 2'- and 3'-deoxyadenosine. The modified species were tested as substrates for activation by their cognate aminoacyl-tRNA synthetases and for misacylation with phenylalanine by yeast phenylalanyl-tRNA synthetase. E. coli alanyl- and lysyl-tRNA synthetases normally aminoacylate their cognate tRNA's exclusively on the 3'-OH group, while yeast phenylalanyl-tRNA synthetase utilizes only the 2' position on its own tRNA. Therefore, the finding that the phenylalanyl-tRNA synthetase activated only those modified tRNAAla and tRNALys species terminating in 3'-deoxyadenosine indicated that the position of aminoacylation in this case was specified entirely by the enzyme, an observation relevant to the more general problem of the reason(s) for using a particular site for aminoacylation and maintaining positional specificity during evolution. Initial velocity studies were carried out using E. coli tRNAAla and both alanyl- and phenylalanyl-tRNA synthetases. As noted in other cases, activation of the modified and unmodified tRNA's had essentially the same associated Km values, but in each case the Vmax determined for the modified tRNA was smaller.     

Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase.

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between 'strong' and 'weak'. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.