A study of the quenching of the intrinsic fluorescence of succinyl-CoA synthetase from Escherichia coli by acrylamide, iodide, and coenzyme A

Abstract

Escherichia coli succinyl-CoA synthetase (SCS) contains three tryptophan residues per mole of alpha beta dimer, and all of them are on the beta subunit. SCS shows an emission maximum at 335 nm which is shifted to 350 nm upon denaturation by urea or guanidine hydrochloride. Acrylamide is able to quench the tryptophan fluorescence in SCS by static and dynamic mechanisms. Substrates give protection against quenching by acrylamide. Binding of ATP to the alpha subunit which has no tryptophans gives as large an effect on the quenching by acrylamide as the binding of coenzyme A (CoA) to the beta subunit. Addition of CoA eliminates the curvature observed in Stern-Volmer plots for acrylamide quenching obtained by lifetime measurements. Potassium iodide does not quench the SCS fluorescence in the presence of CoA. These results suggest that there are heterogeneously emitting tryptophan residues in SCS that are located at the alpha beta subunit contact region close to the CoA binding site. Hence, the tryptophan residues can act as intrinsic reporters of events taking place at the active site of this enzyme. Further, the present results support models for SCS that put the active site at the alpha beta subunit contact region.

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

“…We have shown previously that substrates, such as ATP-Mg2+ and CoA, have a significant effect on the tryptophan fluorescence of E. coli succinyl-CoA synthetase (Prasad et al, 1983a). These observations were consistent with the idea that at least one of the three tryptophan residues is close to or at the active site of the enzyme.…”

“…ATP-Mg2+, succinate, and desulfo-CoA (analogue of Co A), added at saturating concentrations singly, in pairs, and all together, failed to protect the enzyme against modification by A-bromosuccinimide. This was a puzzling result, since quenching of enzyme tryptophan fluorescence by acrylamide has been shown to be affected by the presence of substrates (Prasad et al, 1983a).…”

“…Pearson and Bridger observed that, while the -subunit is capable of autophosphorylation by ATP, the presence of the /8-subunit is required for optimal phosphorylation rates. Recent studies reported from this laboratory indicate that all six tryptophan residues are contained in the two /3-subunits (Prasad et al, 1983a). In addition, the fluorescence properties of at least one of these tryptophans in each /3-subunit is affected significantly by the binding of CoA and ATP to the enzyme.…”

Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum

“…On the basis of the results presented here and other studies Shanmugasundaram et al, 1988), it appears that CoA binds to CODH at a site involving exposed tryptophans which are located at or near the Ni-Fe-C center. Tryptophan residues are shown to be present at the subunit contact region close to the CoA binding site of succinyl-CoA synthetase (Prasad et al, 1983). However, in contrast to our results, subsequent NBS modification studies of the synthetase in the presence of CoA analogue (Ybarra et al, 1986) have shown that the tryptophan residue essential for the catalytic activity is not present at the CoA binding site.…”

Hyaluronidase Activity of Human Hyal1 Requires Active Site Acidic and Tyrosine Residues

“…After washing with 10 column volumes of Tris-buffered saline, Hyal1-FLAG was eluted from the column with FLAG peptide and dialyzed against hyaluronidase assay buffer (50 mM sodium formate, pH 4.0, 150 mM NaCl). Folded structure of mutants that lacked activity was implied by soluble secretion, but also by their intrinsic tryptophan fluorescence profiles (31), which were identical to those of wild-type Hyal1, and red-shifted comparably upon denaturation with 3 M guanidine-HCl (supplemental Fig. S1).…”