Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ATP Synthetase Complexes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed1984

Standardization of parameters for the mycobacillin synthetase activity.

An effective method of preparation involving sonication was developed for cell-free mycobacillin synthetase from Bacillus subtilis. The enzyme showed optimum activity at a buffer concentration of 50 mM (Tris-HCl) and pH 7.5. ATP and Mg2+ which were essential for synthesis showed an optimum requirement at a ratio of 1:1. The synthetase was markedly inhibited by ADP whereas AMP was without any effect. ATP or ATP-generating system could not be replaced by GTP, UTP or CTP. Co2+ and Mn2+ could to some extent substitute Mg2+. Mercapto reagents inhibited the antibiotic synthesis. Exogenous addition of pantothenic acid had no effect.

Bacillus subtilis↗
PubMed2000

Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates.

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein.

Adenosine Monophosphate↗
PubMed1991

An extended dynamic model of oxidative phosphorylation.

The presented model based on an earlier one (Korzeniewski, B. and Froncisz, W. (1989) Studia Biophys. 132, 173-187) simulates concentration changes in time of chemical compounds and thermodynamic forces during respiration of cell suspension in a closed chamber. A set of differential equations solved numerically describes the utilization of oxygen up to anaerobiosis and the behaviour of the system after a sudden pulse of oxygen. Flux control coefficients for most important reactions (enzymes) of oxidative phosphorylation were calculated. A good qualitative and (when a direct comparison is possible) quantitative agreement with experimental results can be observed. The following conclusions can be drawn from the simulation: (1) Wilson's steady state model is not in contradiction with sharing of the control over the respiration between some steps and displacement of the ATP/ADP carrier from equilibrium. (2) The overshoot characteristics of the delta microH+ time-course after reoxygenation can be explained without using the lag-phase kinetics of ATP-synthetase. (3) A 'hot region' (sharp changes of many parameters) can be distinguished when the oxygen concentration approaches zero; only cytochrome oxidase is clearly sensitive on oxygen concentration in all its range. (4) Control over oxidative phosphorylation is shared mainly between inputs of the system (ATP utilization and substrate dehydrogenation) and the proton leak.

Adenine Nucleotides↗
PubMed1983

Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation.

Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.

Amino Acid Sequence↗
PubMed1990

On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations.

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.

Escherichia coli↗
PubMed1982

Glutamine synthetase from ovine brain is a manganese(II) enzyme.

The activation of ovine brain glutamine synthetase by Mn(II) or Mg(II) was studied by steady-state kinetics. The metal ion concentration was varied at several fixed concentrations of ATP, and vice versa, and the resultant kinetic curves were analyzed according to the method of London and Steck [London, W. P., & Steck, T. L. (1969) Biochemistry 8, 1767-1779]. The data for Mg(II) indicated optimal activation at Mg:ATP = 2:1, whereas that for Mn(II) occurred at Mn:ATP = 1:1. This was interpreted as indicating formation of Mg . E . Mg . ATP as the subunit complex of optimum activity with Mg(II) (pHopt 7.5). With Mn(II) (pHopt 5.0), the complex of optimum activity may be either E . Mn . ATP or Mn . E . Mn . ATP, where the Mn . E complex is very tight. So that the latter two possibilities could be distinguished, titrations of the enzyme with Mn(II) were performed, electron paramagnetic resonance being used to determine free Mn(II). Four moles of Mn(II) ions was found to bind per mole of octameric enzyme, with an apparent Kd congruent to 0.54 microM. Addition of either HCl or Nd(III) ions to Chelex-treated enzyme results in the release of 3.7 +/- 0.2 Mn(II) ions. Thus, it appears that four Mn(II) are very tightly bound per octamer and that four more Mn(II) can bind tightly. Neither Mg(II) nor Ca(II) at 50 mM can displace Mn(II) from the Mn4 . E complex, but Mn(II) still binds tightly to apoenzyme or Mn4E in the presence of 50 mM Mg(II). As one proceeds from apo-E to Mn4 . E to Mn4 . E . Mn4 (+/- ATP), the intensity of the fluorescence emission of protein tryptophan residues decreases strongly and successively. The specific activities of the apo-E, Mn4 . E, and Mn4 . E . Mn4 complexes were found to be 0, 50, and 200 units/mg, respectively. If apoenzyme is added to a continuous coupled assay system with Mg(II), buffer, and with or without Mn(II) present, a time-dependent activation is observable with t1/2 congruent to 0.5-1.0 min. The total intracellular concentration of Mn(II) in ovine brain tissue was determined to be 1.9-2.6 microM, whereas the free [Mn(II)] was below 0.5 microM. Since the enzyme binds Mn(II) in preference to other divalent ions, it appears that the enzyme may exist as a manganoenzyme in vivo.

Adenosine Triphosphate↗
PubMed2003

Induced fit and kinetic mechanism of adenylation catalyzed by Escherichia coli threonyl-tRNA synthetase.

Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.

Adenine Nucleotides↗
PubMed1999

X-linked adrenoleukodystrophy: genes, mutations, and phenotypes.

X-linked adrenoleukodystrophy (X-ALD) is a complex and perplexing neurodegenerative disorder. The metabolic abnormality, elevated levels of very long-chain fatty acids in tissues and plasma, and the biochemical defect, reduced peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity, are ubiquitous features of the disease. However, clinical manifestations are highly variable with regard to time of onset, site of initial pathology and rate of progression. In addition, the abnormal gene in X-ALD is not the gene for VLCS. Rather, it encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transmembrane transporter superfamily of proteins. The X-ALD protein (ALDP) is closely related to three other peroxisomal membrane ABC proteins. In this report we summarize all known X-ALD mutations and establish the lack of an X-ALD genotype/phenotype correlation. We compare the evolutionary relationships among peroxisomal ABC proteins, demonstrate that ALDP forms homodimers with itself and heterodimers with other peroxisomal ABC proteins and present cDNA complementation studies suggesting that the peroxisomal ABC proteins have overlapping functions. We also establish that there are at least two peroxisomal VLCS activities, one that is ALDP dependent and one that is ALDP independent. Finally, we discuss variable expression of the peroxisomal ABC proteins and ALDP independent VLCS in relation to the variable clinical presentations of X-ALD.

Adrenoleukodystrophy↗
PubMed1993

In vitro biosynthesis of peptolide SDZ 90-215 by a 1.2 MDa multienzyme polypeptide.

An enzyme fraction with catalytic activities for the biosynthesis of the pipecolic acid containing cyclopeptolide SDZ 90-215 was partially purified and characterized from the genus Septoria. The crude cell homogenate was subjected to polyethyleneimine precipitation, ammonium sulfate precipitation and FPLC gel filtration. The denatured enzyme shows an apparent molecular mass of 1.2 MDa in 3% SDS-PAGE. Peptolide SDZ 90-215 synthetase catalyzes the ATP-PPi exchange reaction dependent on all substituent amino and hydroxy acids. SDZ 90-215 synthetase synthesizes the peptolide in vitro when incubated together with D-lactic acid, all constitutive amino acids in their N-unmethylated form, ATP, magnesium chloride and S-adenosyl-L-methionine. The yield of SDZ 90-215 is higher when O-methyl-L-tyrosine instead of L-tyrosine is used, indicating that O-methylation of tyrosine is not carried out by the synthetase.

Amino Acid Sequence↗
PubMed1977

Glutamine synthetase from pig brain: binding of adenosine triphosphate.

Glutamine synthetase was purified to homogeneity from fresh brain homogenates by a procedure that makes use of the affinity of this ATP requiring ligase to Cibacron 3G-A Blue-Sephadex G-75. It is shown that the interaction of pig brain enzyme with the dye does not concern the active centre but a non-catalytic although specific ATP binding site. Both sides contain a cysteine residue which may react with N-ethylmaleimide. The unprotected succinimidated enzyme is inactive, and aggregates. ATP alone protects only against aggregation, not against inactivation; the ATP/Mg2 complex also hinders inactivation. A tryptic heptadecapeptide isolated from the derivatized enzyme representing the carboxy terminal seems to belong to the active centre; its amino acid sequence was partially determined.

Adenosine Triphosphate↗
PubMed2000

Movement of the biotin carboxylase B-domain as a result of ATP binding.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-A resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-A resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45(o) of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.

Adenosine Triphosphate↗
PubMed1982

Kinetic anticooperativity in pre-steady-state formation of tryptophanyl adenylate by tryptophanyl-tRNA synthetase from beef pancreas. A consequence of the tryptophan anticooperative binding.

The kinetics of formation of tryptophanyl adenylate by tryptophanyl-tRNA synthetase from beef pancreas has been followed by stopped-flow, using the quenching of fluorescence of the enzyme linked to the amino acid activation reaction. Both subunits of this alpha 2 enzyme catalyze the adenylate formation. At saturation with substrates the rate constant of the activation reaction is the same for both subunits. The same behaviour is observed for the pyrophosphorolysis reaction. Both subunits exhibit the same affinity for ATP-Mg in the forward reaction and the same affinity for magnesium pyrophosphate in the backward reaction. On the contrary the formation of tryptophanyl adenylate follows biphasic kinetics when tryptophan concentration is much below saturation. This is independent of ATP-Mg concentration and is the consequence of different affinities of the two subunits for tryptophan as already observed by Graves et al. (1979, Eur. J. Biochem. 96, 509-518) in equilibrium dialysis experiments. A monoadenylate-enzyme complex on one subunit has been prepared. This complex made possible the study of the formation of the second adenylate on the other subunit. The formation of this second adenylate followed first-order kinetics at all ATP-Mg and tryptophan concentrations. The tryptophan concentration dependence of the rate of formation of this second adenylate leads to a Michaelis constant close to the dissociation constant of the low affinity tryptophan site of the enzyme. No isomerization step could be evidenced. The experiments were carried out under two conditions corresponding to those used by Merault et al. (1978. Eur. J. Biochem. 87, 541-550) in the steady state of the tRNATrp aminoacylation reaction (10 mM total magnesium in 100 mM KCl and 1 mM free magnesium ions, both at pH 8.0.25 C). No great difference either in the mechanism or in the dissociation and rate constants was observed but an inhibitory effect of KCl. It is concluded that the enzyme is symmetrical as far as the ATP-Mg and the magnesium pyrophosphate sites are concerned and that the rate of the activation reaction reflects the anticooperative occupancy of the tryptophan sites carried by the two subunits.

Adenosine Monophosphate↗
PubMed1978

The oxidation of long-chain unsaturated fatty acids by isolated rat liver mitochondria as a function of substrate concentration.

1. The oxidation of linoleate by rat-liver mitochondria has been studied as a function of substrate concentration. The oxidation of other long-chain unsaturated fatty acids shows similar characteristics. 2. At low concentrations, linoleate is readily oxidized in the absence of carnitine. Its rate of activation by the intramitochondrial acyl-CoA synthetase (EC 6.2.1.2) and subsequent oxidation is limited by the availability of intra-mitochondrial ATP. 3. A gradual increase of the linoleate concentration leads to (i) a strong depression of the rate of linoleate oxidation, and (ii) uncoupling of respiratory-chain phosphorylation together with induction of a mitochondrial ATPase activity. At still higher linoleate concentrations this ATPase activity is lowered rather than further stimulated and, concomitantly, the rate of linoleate oxidation increases again. 4. Evidence is presented that the inhibition by linoleate of the ATPase activity occurs at the level of the ATPase complex itself. This oligomycin-like effect of linoleate allows intramitochondrial linoleate activation to take place at the expense of ATP derived from substrate-level phosphorylation. 5. At very high concentrations of linoleate, its detergent action predominates and causes a complete inhibition of respiration as well as an extensive stimulation of an oligomycin-insensitive, Mg2+-dependent ATPase activity. 6. Measurement of the binding of radioactively labelled linoleate by isolated mitochondria shows that, at a given ratio of linoleate to mitochondrial protein, the ratio of bound to added linoleate is dependent on the concentration of the mitochondria.

Adenosine Triphosphatases↗
PubMed2004

Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues.

S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.

Adenylyl Imidodiphosphate↗
PubMed1970

Association of ATP: glutamine synthetase adenylyltransferase activity with the P1 component of the glutamine synthetase deadenylylation system.

Regulation of glutamine synthetase (EC 6.3.1.2) in Escherichia coli is mediated by adenylylation and deadenylylation of the enzyme. The present studies show that one protein is a common component of both the adenylylation and deadenylylation systems. Thus, the ATP:glutamine synthetase adenylyltransferase, which catalyzes adenylylation of glutamine synthetase, and one of the two proteins required for deadenylylation (the P(I) protein) are inseparable by a variety of fractionation procedures. The adenylyltransferase and P(I)-deadenylylating activities behave as a single protein upon filtration through Agarose A 0.5 gel, and during chromatography on DE32 cellulose and hydroxyapatite columns. They migrate as a single protein band during electrophoresis on polyacrylamide gel and have identical susceptibilities to heat inactivation. These data indicate that the adenylyltransferase and the P(I)-deadenylylation activity are associated with the same protein complex.

Adenine Nucleotides↗
PubMed1996

Total Glutamine Synthetase Activity during Soybean Nodule Development Is Controlled at the Level of Transcription and Holoprotein Turnover.

Gln synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to yield Gln. In higher plants GS is an octameric enzyme and the subunits are encoded by members of a small multigene family. In soybeans (Glycine max), following the onset of N2 fixation there is a dramatic increase in GS activity in the root nodules. GS activity staining of native polyacrylamide gels containing nodule and root extracts showed a common band of activity (GSrs). The nodules also contained a slower-migrating, broad band of enzyme activity (GSns). The GSns activity band is a complex of many isozymes made up of different proportions of two kinds of GS subunits: GSr and GSn. Root nodules formed following inoculation with an Nif- strain of Bradyrhizobium japonicum showed the presence of GS isoenzymes (GSns1) with low enzyme activity, which migrated more slowly than GSns. Gsns1 is most likely made up predominantly of GSn subunits. Our data suggest that, whereas the class I GS genes encoding the GSr subunits are regulated by the availability of NH3, the class II GS genes coding for the GSn subunits are developmentally regulated. Furthermore, we have demonstrated that the GSns1 isozymes in the Nif- nodules are relatively more labile. Our overall conclusion is that GSns activity in soybean nodules is regulated by N2 fixation both at the level of transcription and at the level of holoprotein stability.

Journal Article↗
PubMed1975

Guanosine monophosphate synthetase from Escherichia coli B-96. Inhibition by nucleosides.

The mechanism of inhibition of GMP synthetase by purine and purine-analog nucleosides was investigated. It was found that in addition to allowing the nucleoside to bind to the enzyme (Udaka, S., and Moyed, H. S. (1963) J. Biol. Chem. 238, 2797)PPi was also a competitive inhibitor with respect to ATP. A rate equation was derived to describe this inhibitory model for two competitive inhibitors where the binding of one inhibitor is contingent upon the binding of the other. The inhibition constants for a large number of nucleosides were then determined. It was found that the initial enzyme-inhibitor complex (of all nucleoside inhibitors) was slowly (0.2 min-1) transformed into a secondary (nondissociating) complex. The two inhibitory complexes appeared to exist in equilibrium. While decoyenine, N6-allyladenosine, and adenosine had similar inhibition constants for the initial complex (0.7 to 1.0 muM), their apparent inhibition constants for the secondary complex were 0.004, 0.06, and 0.5 muM respectively. These differences in the apparent dissociation constants from the secondary complexes are due to different equilibria between the initial and the secondary complexes. The ratios of the secondary complex to the initial complex at equilibrium were 3,250, 290, and 11 for decovenine, N6-allyladenosine, and adenosine, respectively.

Adenosine↗
PubMed1997

The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in bakers' yeast.

An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375,000-400,000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzes a reaction between 4'-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase, to reform CoA. This sequence of reactions, referred to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by which the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment segment is referred to as the 10,000-15,000 M(r) subunit. The 5'-ADP-4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthetase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10,000-15,000 M(r) subunit. Thus, the 10,000-15,000 M(r) subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.

Catalysis↗