The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes. The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex. The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read-through transcript that extends from the suc promoter to a potential rho-independent terminator at the distal end of sucD. The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes. The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase. The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase. The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme. This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit.

Primary structure of the succinyl-CoA synthetase of Escherichia coli

Photoaffinity-labeling peptide substrates for farnesyl-protein transferase and the intersubunit location of the active site.

Succinyl‐CoA Synthetase Structure‐Function Relationships and Other Considerations

Publisher Summary This chapter focuses on the characterization of phosphorylated target protein by phosphorus-31 nuclear magnetic resonance (31P NMR). It describes the preparation of a sample of phosphorylated protein for analysis by 31P NMR. 31P NMR can be used to assign the type of amino acid residue the phosphate is covalently attached to, such as the β-hydroxyl of a serine or threonine residue, or the N-1 or N-3 nitrogen of the imidazole ring of a histidine. 31P NMR can provide a range of information about the nature and dynamics of the phosphorylation site that cannot be obtained by other methods. The greatest strength of the NMR method is that if two or more phosphorylation sites exist on a protein, each phosphoryl group can be individually monitored by 31P NMR. 31P NMR has several important practical advantages. It is a nondestructive spectroscopic method relying on the absorption of very low radiofrequency irradiation. Thus, the sample can be recovered intact after NMR analysis. The NMR experiment can be done under the conditions of temperature and pH that are favorable for the stability of the phosphorylated protein.

Phosphorus-31 nuclear magnetic resonance studies of phosphorylated proteins

31P NMR has been used to investigate the nature of the two chemically distinct phosphorylation sites of ATP-citrate lyase from rat liver. The "regulatory" or "structural" phosphorylation site is acid stable and known to be phosphoserine. The "catalytic" site is very acid labile and has been suggested by different workers to contain either phosphohistidine or an acyl phosphate group. We have demonstrated the presence of both endogenous phosphoserine and phosphoserine introduced after treatment of the lyase with the catalytic subunit of cAMP-dependent protein kinase. This structural phosphate group could be titrated and was readily removed by alkaline phosphatase; these facts, together with the narrow line width of the 31P NMR signal, suggest that it is relatively mobile and located near the surface of the protein. 31P NMR spectra of ATP-citrate lyase that had previously been exposed to fairly high concentrations of potassium chloride (1.5 M), or that had been denatured in detergent and 2-mercaptoethanol, clearly identified phosphohistidine as the catalytic phosphate group. That phosphohistidine is indeed a catalytic intermediate was demonstrated by the disappearance of the resonance in the presence of the substrates citrate and coenzyme A. The line width of the phosphohistidine resonance indicated that the catalytic phosphohistidine residue has negligible residual mobility on the protein. These results are consistent with the pattern of earlier observations on the chemical environments of phospho groups that serve a regulatory or structural role as opposed to a catalytic function in proteins.

Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase

A phosphorus 31 nuclear magnetic resonance study of the intermediates of the Escherichia coli succinyl coenzyme A synthetase reaction. Evidence for substrate synergism and catalytic cooperativity.

H J Vogel

Papers

A phosphorus 31 nuclear magnetic resonance study of the intermediates of the Escherichia coli succinyl coenzyme A synthetase reaction. Evidence for substrate synergism and catalytic cooperativity.