Cloning, characterization, and expression of the β subunit of pig heart succinyl‐CoA synthetase

Bailey, D.; Wolodko, William T.; Bridger, William A.

doi:10.1002/pro.5560020808

Cited by 19 publications

(12 citation statements)

References 21 publications

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“…The design of the gene is different from one used earlier (37) because the crystal structure showed that a methionine residue inserted before the first residue of the mature ␤-subunit could interfere with catalysis (36). It may be the reason for the reduced specific activity of the enzyme produced in E. coli (37). Modified cDNAs encoding the mature ␣-and ␤-subunits were cloned into a single expression plasmid, each cDNA being placed downstream of a separate T7 promoter.…”

Section: Methodsmentioning

confidence: 99%

Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Fraser

Hayakawa

Hume

et al. 2006

Journal of Biological Chemistry

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Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 Å resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20␤) and with backbone atoms provide the specificity. The ␥-phosphate interacts with the side chain of an arginine residue (Arg-54␤) and with backbone amide nitrogen atoms, leading to tight interactions between the ␥-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the ␥-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the ␣-and ␤-phosphates allow the ␥-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the ␤-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.The enzyme succinyl-CoA synthetase (SCS) 4 uses ATP or GTP to catalyze the formation of succinyl-CoA from succinate and coenzyme A. In animals, two different isoforms exist, one specific for ATP and the other specific for GTP (1). The two isoforms include the same ␣-subunit, but different ␤-subunits (2). The amino-terminal domain of the ␤-subunit has an ATP-grasp fold (3-5), and Mg 2ϩ -ADP was shown to bind in this domain of the Escherichia coli SCS using labeling experiments and site-directed mutagenesis (6) and by soaking the nucleotide into crystals and determining the structure of the resulting complex (7). The ATP-grasp fold has been found in a number of other enzymes (8) (24), and RNA ligase 2 (25). The determinations of the structures of several of these enzymes in complex with nucleotides, nucleotide analogues, and their other substrates has led to a good understanding of the interactions that are important in their binding and catalysis.The biological roles of the ATP-and GTP-specific SCS have not been fully delineated. Originally it was thought that the primary role for SCS was in the citric acid cycle, where it was responsible for the breakdown of succinyl-CoA to succinate and coenzyme A accompanied by the phosphorylation of nucleotide diphosphate to nucleotide triphosphate (26). This step provides the only substrate-level phosphorylation of the citric acid cycle. It was thought that some species had SCS that could use either ADP or GDP, e.g. E. coli (27), while others had SCS that coul...

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Section: Methodsmentioning

confidence: 99%

Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Fraser

Hayakawa

Hume

et al. 2006

Journal of Biological Chemistry

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“…As described under Table II. The sequences are for the mature proteins, assuming that the signal peptides are cleaved at the same position as in G-␤ in pig (22).…”

Section: Sequences Of the ␤-Subunits Of G-scs And A-scs In Highermentioning

confidence: 99%

“…Earlier suggestions that the ␣-subunit is more highly conserved than the ␤-subunit (8,13,22) were based on comparisons of sequences of ␤-subunits that we now know to vary in their specificity. When ␤-subunits of the same specificity are compared, identity of sequence is as high as found in ␣-subunits.…”

Section: Figmentioning

confidence: 99%

“…Extensive work on the genes encoding SCS have been carried out on the ATP-preferring enzyme in E. coli (20) and the GTPspecific enzyme in pig (21,22). These studies, together with the completed genome projects for several procaryotes and yeast, show that the ␣-and ␤-subunits are products of separate genes.…”

mentioning

confidence: 99%

“…An alternative splicing mechanism for the ␣-subunit in pig generates two isoforms of SCS that are GTP-specific (24). Prior to the work described here, the only sequence reported for the ␤-subunit of SCS in a vertebrate species was for G-SCS from pig (22).…”

mentioning

confidence: 99%

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Genetic Evidence for the Expression of ATP- and GTP-specific Succinyl-CoA Synthetases in Multicellular Eucaryotes

Johnson

Mehus

Tews

et al. 1998

Journal of Biological Chemistry

137

144

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Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

Solow¹,

Bischoff²,

Zylka³

et al. 1998

Protein Science

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When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [Y-'~P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35-and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the a-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-ThrAsp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.Keywords: Archaea; Archaebacteria; hexosephosphate mutase; phosphoprotein; succinyl-CoA synthetase Phosphate represents one of nature's most versatile and important chemical moieties (Westheimer, 1987). It comprises (1) the basic unit for the cell's energy currency, ( 2 ) a key chemical activator of organic functional groups in biocatalysis, (3) a charge-rich anchor for confining biomolecules within the cell membrane, (4) the linker that holds together the polymers that encode genetic information, and (5) a molecular switch for modulating the functional properties of key proteins. Although our general appreciation of the importance of phosphate in living organisms extends to the members of the Archaea-the third, most recent, phylogenetic domain to be recognized (Olsen & Woese, 1993)-in many areas, specific information is lacking. This is particularly true with regard to the interactions of phosphate with archaeal proteins. Only recently have archaeal phosphoproteins begun to be identified. The first was a CheA-like histidine kinase that functions as part of a twocomponent signal transduction cascade in Halobacterium salinarium Reprint requests to: Peter J. Kennelly, Department of Biochemistry, Virginia Polytechnic Institute and State University, 123 Engel Hall, Blacksburg, Virginia 24061-0308; e-mail: pjkennel@vt.edu. (Rudolph & Oesterhelt, 1995). The second was a methyltransferase activation protein from Methanosarcina...

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Cloning, characterization, and expression of the β subunit of pig heart succinyl‐CoA synthetase

Cited by 19 publications

References 21 publications

Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Genetic Evidence for the Expression of ATP- and GTP-specific Succinyl-CoA Synthetases in Multicellular Eucaryotes

Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

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