Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

Abstract

Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinylcoenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and P i are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.The processes of cell differentiation, growth, and aging reflect the selection of specific pathways to be activated or inactivated. Such metabolic regulation may occur at the level of expression of components in those pathways or by modulating their activities. We have been investigating how changes in energy metabolism reflect or determine the cell's genetic potential for growth and development. In particular, our studies have focused on the regulation of succinyl-coenzyme A (CoA) synthetase (SCS), the only enzyme in the citric acid cycle that catalyzes a substrate-level phosphorylation reaction. This reaction is completely reversible, allowing for the production of nucleoside triphosphate (NTP) during aerobic metabolism and the synthesis of succinyl-CoA for anabolic reactions. Partial reactionswhere NDP is nucleoside diphosphate and E is the enzyme. As seen in the schema above, the phosphoenzyme is an intermediate in the reaction and can be formed by autophosphorylation with either an NTP (top partial reaction) or succinyl-CoA and P i . The enzyme is composed of two distinct protein subunits, ␣ and ␤. There are two known forms of the enzyme. In gram-negative bacteria such as Escherichia coli, the enzyme is a tetramer of two ␣ and two ␤ subunits. It preferentially uses adenine nucleotides and will be referred to here as the A-form enzyme. G-form enzymes, which appear to use exclusively guanine nucleotides in the reaction, predominate in eukaryotes and gram-positive bacteria. They function as dimers composed of one ␣ and one ␤ subunit. The physiological significance of the differences in quaternary structure and nucleotide preferences with respect to enzyme action or organization in the citric acid cycle is unknown. In either case, it is a histidine residue in the ␣ subunit that is phosphorylated to form the phosphoenzyme intermediate. (for reviews, see references 5 and 14).In the presence of saturating or near-saturating concentrations of substrates, SCSs f...

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