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.
Bridger
W A
WA
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Wolodko
W T
WT
Henning
W
W
Upton
C
C
Majumdar
R
R
Williams
S P
SP
D000818:Animals; D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000096:biosynthesis; D008928:Mitochondria / Q000201:enzymology; D013389:Succinate-CoA Ligases / Q000096:biosynthesis
D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases