Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Abstract

Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon

“…during anaerobiosis. The enzyme is assembled as a nondissociating tetramer (a2,82) from two types of subunit: the a-subunit (Mr 29600), which contains the histidine residue that is phosphorylated during catalysis and the ATP binding site; and the ,-subunit (Mr 41400), which contains the binding sites for succinate and CoA (Bridger, 1974;Nishimura, 1986). The corresponding enzymes from eukaryotes and Gram-positive bacteria differ in being non-associating dimers (cfl) with different nucleotide specificities (Weitzman, 1981;Wolodko et al, 1986).…”

“…The corresponding enzymes from eukaryotes and Gram-positive bacteria differ in being non-associating dimers (cfl) with different nucleotide specificities (Weitzman, 1981;Wolodko et al, 1986). Chemical modification studies (with the E. coli enzyme) have indicated important functional roles for two cysteine residues, a tryptophan residue and one histidine residue in addition to the phosphorylation site (Nishimura, 1986), and the enzyme has been crystallized for structure determination (Wolodko et al, 1984).…”

“…There is evidence from chemical modification studies that a second histidine residue is important for stabilizing the reactive thiol form of a cysteine residue in the CoA binding site of the /3 subunit (Collier & Nishimura, 1979). It has also been found that the enzyme is inactivated by modification of one of the three tryptophan residues in the , subunit (Ybarra et al, 1986). Inspection of the primary structure revealed several putative CoA binding sites (Buck et al, 1985), and it may be significant that two of these are associated with residues of the putative active site types:…”

Regulation of Human Carbonic Anhydrase I (hCAI) Activity by Using a Photochromic Inhibitor

“…In biology, enzymatic control is typically achieved through the use of allostery [1] or by covalently modifying the enzyme (by phosphorylation or dephosphorylation, for example). [2] Some of the original attempts to artificially influence the activity of enzymes rely on chemical modifications of the enzyme structure, [3][4][5][6] an approach that is limited by the fact that the regulation is not reversible. The use of light as a stimulus offers a heightened level of control, and photoresponsive compounds would provide the reversibility needed for practical use.…”

Regulation der Aktivität von humaner Carboanhydrase I (hCAI) durch einen photochromen Inhibitor

“…Erste Versuche zur künstlichen Regulation der Enzymaktivität basierten auf chemischer Veränderung der Enzymstruktur. [3][4][5][6] Diese Art der Regulation ist jedoch nicht reversibel. Licht als Stimulus ermöglicht eine einfache Kontrolle, und durch photochrome Verbindungen kann Reversibilität erreicht werden, was für praktische Anwendungen wichtig ist.…”