Intrinsic fluorescence of succinyl-CoA synthetase and four tryptophan mutants. Tryptophan 76 and tryptophan 248 of the beta-subunit are responsive to CoA binding.
Previous studies showed that modification of an average of one of the three tryptophan residues of succinyl-CoA synthetase of Escherichia coli abolished enzyme activity, but did not prevent phosphorylation of the enzyme by ATP [Ybarra, J., Prasad, A. R. S., & Nishimura, J.S. (1986) Biochemistry 25, 7174-7178]. In the present study, single mutations in which each of the three tryptophans (beta-Trp43, beta-Trp76, and beta-Trp248) has been changed to phenylalanine (designated W43F, W76F, and W248F) have been accomplished by the technique of site-directed mutagenesis and the mutant proteins isolated. In addition, a double mutant in which beta-Trp43 and beta-Trp248 were changed to phenylalanines (W43,248F) has also been isolated. Each of the mutant enzymes was practically as active as wild type. Since the emission spectrum of beta-Trp76 reflected a low fluorescence intensity for this residue, it was possible to obtain the emission spectrum of each tryptophan residue by using W43F, W248F, and W43,248F. From the positions of the emission maxima and the results of iodide quenching of fluorescence, it was deduced that beta-Trp248 is a surface residue, beta-Trp43 is buried, and beta-Trp76 is intermediate in location. Coenzyme A, but no other substrate, protected the fluorescence of beta-Trp76 and beta-Trp248, but not of beta-Trp43, against quenching by acrylamide. These results are consistent with an interaction between beta-Trp76 and beta-Trp248 and the binding site for CoA.
Nishimura
J S
JS
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Mann
C J
CJ
Ybarra
J
J
Mitchell
T
T
Horowitz
P M
PM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics*; D007454:Iodides / Q000494:pharmacology; D009154:Mutation; D010649:Phenylalanine; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013389:Succinate-CoA Ligases / Q000235:genetics* / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics
D007454:Iodides; D013386:Succinates; D010649:Phenylalanine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D003065:Coenzyme A