Fluorescence energy transfer measurements of spatial relationships between sulfhydryl groups of thiolase I from porcine heart.
Mitochondrial thiolase I from pig heart has been found to have at least two and possibly three reactive sulfhydryl residues at or near the active site [Izbicka-Dimitrijević, E., & Gilbert, H. F. (1982) Biochemistry 21, 6112-6118; Izbicka-Dimitrijević, E., & Gilbert, H. F. (1984) Biochemistry 23, 4318-4324]. In the native enzyme, fluorescein mercuric acetate reacts with two of the sulfhydryl groups and inactivates the enzyme with a rate constant of 1.6 X 10(-4) M-1 s-1 in 0.1 M Tris-acetate, pH 7.0. The presence of saturating (250 microM) concentrations of acetoacetyl coenzyme A protects against both modification and inactivation. The acetyl enzyme, a normal intermediate in the reaction catalyzed by thiolase, is not inactivated by fluorescein mercuric acetate although one sulfhydryl group out of five per mole of thiolase subunit is still available for reaction with the reagent. Fluorescein mercuric acetate and S-mercurio-N-dansyl-L-cysteine (Dns-Cys-SHg+) have been used to differentially label two of the sulfhydryl groups in thiolase. The distance between Dns-Cys-SHg+ (donor) and fluorescein mercuric acetate (acceptor) determined by the fluorescence energy transfer is less than 14 A. The fluorescent analogue of coenzyme A, 1,N6-etheno coenzyme A, is recognized by thiolase as a substrate (Km = 21 microM); however, substrate inhibition and equilibrium dialysis show that the affinity of the free enzyme for CoA is quite low (Ki = 100 microM). The quantum yield of the fluorescence of the three thiolase tryptophan residues is low (0.024), corresponding to about 12% of the fluorescence expected from equivalent concentrations of tryptophan.
D000101:Acetyl-CoA C-Acetyltransferase; D000123:Acetyltransferases; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D005452:Fluoresceins; D008929:Mitochondria, Heart / Q000201:enzymology*; D009941:Organomercury Compounds; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D013552:Swine
D005452:Fluoresceins; D009941:Organomercury Compounds; D013438:Sulfhydryl Compounds; C001673:fluorescein mercuric acetate; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase