Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum.
The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.
Kurz
Linda C
LC
Department of Biochemistry and Molecular Biophysics, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA. kurz@biochem.wustl.edu
Fite
Brett
B
Jean
John
J
Park
Jung
J
Erpelding
Tim
T
Callis
Patrik
P
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D007700:Kinetics; D062907:Oxaloacetic Acid / Q000737:chemistry; D011487:Protein Conformation; D011789:Quantum Theory; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013379:Substrate Specificity / Q000235:genetics; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D062907:Oxaloacetic Acid; D014364:Tryptophan; D002950:Citrate (si)-Synthase