Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase.

Succinyl-CoA synthetase (EC 6.2.1.5, succinate:CoA ligase (ADP-forming] of Escherichia coli is an alpha 2 beta 2 tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the alpha subunit, and the succinate and CoA binding sites are in beta. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 alpha----Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the beta subunit; this reaction does not involve covalent participation of His-246 alpha. We have found that the His-246 alpha----Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 alpha is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.

Bacterial expression of rat liver succinyl-CoA synthetase alpha-subunit. Factors that contribute to blocked translation of transcripts encoding a mitochondrial signal sequence.

This study comprises a detailed evaluation of factors that are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the alpha-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block the expression of other genes such as E. coli lacZ. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115-nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes.

A detailed structural description of Escherichia coli succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle. A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution. The crystals are of space group P4322, having unit cell dimensions a=b=98.68 A, c=403.76 A and the data set includes the data measured from 23 crystals. E. coli SCS is an (alphabeta)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals. The crystal packing leaves two choices for which pair of alphabeta-dimers form the physiologically relevant tetramer. The copies of the alphabeta-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found. CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the alpha-subunit. The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two alpha-helices, one from the C-terminal domain of the alpha-subunit and the other from the C-terminal domain of the beta-subunit. These two domains have similar topologies, despite only 14 % sequence identity. By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the beta-subunit. If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide.

A dimeric form of Escherichia coli succinyl-CoA synthetase produced by site-directed mutagenesis.

Succinyl-CoA synthetase (SCS) catalyzes the substrate-level phosphorylation step of the citric acid cycle. The enzyme from Escherichia coli is an (alphabeta)2-heterotetramer with two active sites, one in each alphabeta-dimer. To determine whether the two active sites could function independently, mutations were made to split the tetramer into alphabeta-dimers. Because two choices for the tetramer (I and II) were possible from the X-ray crystallographic analyses, mutations were made at two different interfaces. All mutations based on tetramer I resulted in an intact tetramer. Of the two mutants based on tetramer II, one was insoluble and the other, where M156beta, Y158beta, R161beta and E162beta were changed to D, D, E and R, respectively, was a dimer. This quaternary structure was confirmed by fast protein liquid chromatography, blue native PAGE and ultracentrifugation. The DDER mutant has kinetic parameters similar to the tetrameric E. coli enzyme. Like the tetrameric enzyme, it shows ATP-facilitated dethiophosphorylation, proving that this property is a single-site effect. The ATP-facilitated dethiophosphorylation is inhibited by phosphate. It is concluded that dimerization of alphabeta-dimers is not a prerequisite for catalytic competency nor for alternating sites cooperativity in the tetramer. The rationale behind the dimer-of-dimers in E. coli SCS is still not known, but increased solubility, increased stability and in vivo interactions of the tetramer with other proteins are still possibilities.

Phosphorylated and dephosphorylated structures of pig heart, GTP-specific succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes the reversible phosphorylation/dephosphorylation reaction:¿¿¿rm succinyl ¿hbox ¿-¿CoA+NDP+P_i¿leftrightarrow succinate+CoA+NTP¿¿where N denotes adenosine or guanosine. In the course of the reaction, an essential histidine residue is transiently phosphorylated. We have crystallized and solved the structure of the GTP-specific isoform of SCS from pig heart (EC 6.2.1.4) in both the dephosphorylated and phosphorylated forms. The structures were refined to 2.1 A resolution. In the dephosphorylated structure, the enzyme is stabilized via coordination of a phosphate ion by the active-site histidine residue and the two "power" helices, one contributed by each subunit of the alphabeta-dimer. Small changes in the conformations of residues at the amino terminus of the power helix contributed by the alpha-subunit allow the enzyme to accommodate either the covalently bound phosphoryl group or the free phosphate ion. Structural comparisons are made between the active sites in these two forms of the enzyme, both of which can occur along the catalytic path. Comparisons are also made with the structure of Escherichia coli SCS. The domain that has been shown to bind ADP in E. coli SCS is more open in the pig heart, GTP-specific SCS structure.

Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase.

The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active-site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme-CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS-PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C-terminal fragment is blocked at its N-terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N-terminus corresponding to the site of proteolysis and a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active-site structure and catalytic mechanism of CoA transferase.

Cloning, characterization, and expression of the beta subunit of pig heart succinyl-CoA synthetase.

The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alpha beta dimer. Both GTP- and ATP-specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succinyl-CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mitochondrial targeting sequence. The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA synthetase beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were previously cloned (about 45% identical to beta from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429-434), we found no tryptophan residue to be encoded in the sequence for the mature beta subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart beta subunit and its counterpart alpha subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl-CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.

Phosphorylation by cyclic AMP-dependent protein kinase does not affect the association of ATP citrate-lyase with isolated mitochondria.

ATP citrate-lyase is known to be a substrate for various protein kinases, but the functional role, if any, of kinase-directed phosphorylation of this enzyme has not been identified. Recently, Strålfors [(1987) J. Biol. Chem. 262, 11486-11489] has suggested that effects on the association of this enzyme with mitochondria may account for the observed ability of isoproteronol or insulin to promote immobilization of ATP citrate-lyase in permeabilized cells. Here we report studies involving phosphorylation of the pure enzyme in vitro using cyclic AMP-dependent protein kinase. We show that phosphorylation has no significant effect on the fraction of the enzyme that may be bound to isolated mitochondria.

The subunits of succinyl-coenzyme A synthetase--function and assembly.

Succinyl-CoA synthetase is made up of two kinds of subunits, designated alpha and beta. The enzyme from Escherichia coli is an alpha 2 beta 2 tetramer (mol. mass. 142 kDa), whereas the mammalian mitochondrial species is an alpha beta dimer. By means of active enzyme centrifugation, we have shown that the active form of the bacterial enzyme is the tetramer even at very low assay concentrations, while the pig heart enzyme is a non-associating dimer over a wide concentration range. The E. coli enzyme shows distinct half-of-the-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha-subunit that represents a step in catalysis. Many lines of evidence (hybrid enzyme formation, oxygen exchange kinetics, 31P-n.m.r. studies) suggest that co-operative interactions between alternatingly functional active sites on the two halves of the E. coli enzyme contribute to its catalytic efficacy. In further refining this model for catalysis, we have shown that the monothiophosphorylated E. coli enzyme does not catalyse exchange of 18O from the beta, gamma-bridge to the beta-non-bridge position of ATP, indicating that the enzyme does not undergo even transient bis-phosphorylation. As a first step in studying the in vivo synthesis and assembly of the enzyme in the mammalian mitochondrial matrix, we have cloned and sequenced a 900 bp cDNA fragment that encodes most of the alpha subunit of rat liver succinyl-CoA synthetase. The derived amino acid sequence shows an impressive degree of homology to that of the alpha subunit of the enzyme from E. coli. We have shown that the alpha subunit in rat liver is a discrete nuclear gene product, complete with cleavable signal sequence to specify mitochondrial targetting.

Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase.

We have isolated a full-length cDNA clone encoding the cytoplasmic precursor to pig heart mitochondrial CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase, EC 2.8.3.5], a key enzyme for ketone body catabolism. The deduced amino acid sequence indicates the presence of a 39-residue mitochondrial signal sequence and a 481-residue mature protein of molecular weight 52,197. CoA transferase is known to be susceptible to proteolytic cleavage to produce a nicked but active enzyme. We have identified the site of proteolysis, and analysis of the sequence in its vicinity suggests that the polypeptide may fold into two domains connected by a highly hydrophilic bridge.

Probes of the structure of phosphoenolpyruvate synthetase: effects of a transition state analogue on enzyme conformation.

Phosphoenolpyruvate synthetase of Escherichia coli is strongly inhibited by oxalate. The magnitude of the inhibition constant for oxalate suggests that this compound acts to produce a transition state analogue, in keeping with the suggestion of others that oxalate mimics the structure of enolpyruvate, a presumed catalytic intermediate in the enzymatic reaction. The addition of oxalate together with ATP results in a dramatic shielding of sensitive amino acid residues from reaction with both N-ethyl maleimide and phenylglyoxal. Thus, under conditions otherwise giving rise to almost complete inactivation by either reagent, no loss of activity is detectable in the presence of oxalate and ATP. These results indicate the formation of an enclosed structure during catalysis in which reactive groups are rendered quite inaccessible to solvent.

relA Gene control of bacterial glycogen synthesis.

Starvation of Escherichia coli K12 for an amino acid results in the stimulation of bacterial glycogen synthesis in cells containing the relA+ gene, but not in cells carrying the relA- allele. Similarly, a large difference in glycogen content is demonstrable between relA+ and relA- cells in stationary phase. It is concluded that guanosine 5',3' -bis(diphosphate) (ppGPP) or some related relA -dependent metabolite is involved in the regulation of bacterial glycogen synthesis. Detection of significant basal levels of glycogen in a relA- strain of E. coli and in unstarved relA+ C. coli indicates that relA control is not absolutely required for glycogen synthesis but serves as a signal for modulation in response to nutrient availability.

Phosphorus-31 nuclear magnetic resonance pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a.

31P nuclear magnetic resonance (NMR), pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a (in the presence of the inhibitor glucose) show that the resonances for the phosphoserine regulatory sites shift with pH. Analysis of line widths indicates that both residues have considerable mobility. These results are in agreement with studies on similar phosphorylated sites on other proteins, leading us to propose that mobility is a general feature of such regulatory sites. pH titration studies on a series of model compounds have indicated that an empirical correlation exists between the Hill coefficient n (a measure of the cooperativity of the titration curve) and the presence of charged groups in the vicinity of the phosphoryl moiety. Moreover, these studies showed that within one class of similarly substituted phosphorus compounds the chemical shifts, the titration behaviour, and the pKa2 were comparable and allow for easy identification of these compounds. The pKa2 values for phosphoserine residues of proteins are in general slightly higher than those of phosphomonoester-containing small compounds. The titration data prompt our estimation that the maximal amount of energy associated with a salt linkage between a phosphoseryl side chain and a positively charged group is about 5 kcal (1 cal = 4.1868 J).

Mitochondrial translocation and processing of the precursor to the alpha-subunit of rat liver succinyl-CoA synthetase.

Succinyl-CoA synthetase functions in the mitochondrial matrix as an alpha beta-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (lambda SCS19) that apparently encodes the cytoplasmic precursor to the alpha-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature alpha-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific alpha-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.

Positional oxygen isotope exchange as a probe for the mechanism of catalysis by Escherichia coli succinyl coenzyme A synthetase.

Succinyl-CoA synthetase of Escherichia coli has an alpha 2 beta 2 subunit structure. The enzyme shows strict half-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha subunit that represents a step in catalysis. Several lines of evidence indicate that this behavior may result from cooperative interactions between alternatingly functional active sites, so that subsequent steps in catalysis at one site may be promoted by phosphoryl transfer to the site on the neighboring half of the molecule. This study is directed toward learning more about the nature of these cooperative interactions. Here we have used positional isotope exchange (i.e., exchange of 18O between the beta, gamma bridge and the beta nonbridge position of ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was prepared in which one of the two active sites was thiophosphorylated; this species thus has one of its two active-site histidine residues occupied and unavailable for further reaction with ATP. Treatment of this monothiophosphorylated enzyme with [beta, gamma-18O]ATP resulted in no significant scrambling of isotope into the nonbridge position, clearly indicating that the enzyme does not undergo even transient bisphosphorylation. We interpret the results in terms of a model of catalysis in which phosphoryl transfer to the second site occurs in concerted fashion with transfer from the first.

Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits.

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.

Folding and assembly of the Escherichia coli succinyl-CoA synthetase heterotetramer without participation of molecular chaperones.

Succinyl-CoA synthetase of Escherichia coli (alpha 2B2 subunit structure) has been shown to fold and assemble without participation by molecular chaperones. Renaturation experiments showed that purified bacterial chaperone GroEL has no effect on the folding and assembly of the active tetrameric enzyme. When isolated 35S-labeled alpha or beta subunits were incubated with GroEL in the absence of ATP, there was no complex formation between the subunits and GroEL. These in vitro results were confirmed by in vivo analysis of the folding and assembly of newly synthesized succinyl-CoA synthetase subunits. When expression of the subunits was induced in E. coli strains that bear GroEL or GroES temperature-sensitive mutations, the assembly of active succinyl-CoA synthetase was not affected as the temperature was raised to 43 degrees C. These and other observations are discussed that indicate that folding and assembly of succinyl-CoA synthetase may be independent of assistance by any chaperone.

Phosphorus-31 nuclear magnetic resonance studies of the two phosphoserine residues of hen egg white ovalbumin.

Ovalbumin contains two phosphoserine residues that give rise to two well-resolved resonances in a 31P NMR spectrum. Ovalbumin samples that have been digested with a variety of phosphatases may give rise to only one phosphoserine resonance, indicating that one of the two phosphorylated sites is relatively inaccessible for phosphatase action. By comparison of the amino acid sequence of the peptide containing the nonsusceptible phosphate to the overall primary structure, we have assigned the resonances observed (pH 8.3) at 5.0 and 4.75 ppm to phosphoserines-68 and -344, respectively. pH titration behavior and susceptibility of the phosphoserine residues to phosphatases indicate that both are located on the surface of the protein. Both residues have a pKa = 6.00-6.04. Analysis of the Hill coefficients measured for the pH titrations and the JPH coupling constants indicate that neither residue interacts with other charged groups on the surface of the protein. Frequency dependence of 31P NMR parameters shows that at higher magnetic field strengths the contribution of chemical shift anisotropy to the line width becomes very significant. We have calculated from the field-dependent terms that phosphoserine-344 is mobile with respect to the protein surface but that phosphoserine-68 is more restricted in its motion. The latter is also involved in a pH-dependent conformational change, since it is shielded from hydrolysis by phosphatases at higher pH. A comparison of the amino acid sequence of the phosphoserine-68 site shows that it has a striking homology to the active-site peptides of a wide variety of hydrolytic enzymes. Moreover, a comparison with the primary sequences of casein suggests that both proteins are phosphorylated by a protein kinase that specifically recognizes a Ser-X-Glu peptide.

The kinetic properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Initial rate kinetic studies on phosphoenolpyruvate carboxykinase of Escherichia coli are consistent with either of two sequential kinetic mechanisms: rapid equilibrium random, or partially ordered. However, the kinetics of isotope exchange at chemical equilibrium allows clear discrimination in favor of a random mechanism containing a preferred pathway of substrate addition and product release. All plots of exchange rates vs. reactant concentrations leveled off at constant rates at saturating levels of substrates; since complete inhibition of any exchange was not observed, a compulsory sequence may be eliminated. High concentrations of phosphoenolpyruvate and (or) ATP repressed the oxaloacetate in equilibrium bicarbonate exchange, however, indicating that the latter pair of substrates add most favorably to the free enzyme. Exchange rates between various substrate-product pairs were not identical, ruling out a formally rapid equilibrium random mechanism with rate-limiting interconversion of the central complex. A comparison of the relative rates of the overall reaction and the ATP-dependent oxaloacetate in equilibrium bicarbonate exchange showed the latter to be much faster than net formation of oxaloacetate. The requirement for ATP in promoting this exchange may be rationalized in terms of substrate synergism, with occupation of the ATP binding site a requisite for its catalysis.