Fluorescence energy transfer measurements of spatial relationships between sulfhydryl groups of thiolase I from porcine heart

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Abstract
Mitochondrial thiolase I from pig heart has at least 2 and possibly 3 reactive SH residues at or near the active site. In the native enzyme, fluorescein mercuric acetate reacts with 2 of the SH groups and inactivates the enzyme with a rate constant of 1.6 .times. 10-4 M-1 s-1 in 0.1 M Tris-acetate, pH 7.0. The presence of saturating (250 .mu.M) concentrations of acetoacetyl-CoA 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 1 SH group out of 5/mol of thiolase subunit is still available for reaction with the reagent. Fluorescein mercuric acetate and S-mercurio-N-dansyl-L-cysteine (Dns-Cys-SHg+) were used to differentially label 2 of the SH 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 .ANG.. The fluorescent analog of CoA, 1,N6-etheno CoA, is recognized by thiolase as a substrate (Km = 21 .mu.M); however, substrate inhibition and equilibrium dialysis show that the affinity of the free enzyme for CoA is quite low (Ki = 100 .mu.M). The quantum yield of the fluorescence of the 3 thiolase tryptophan residues is low (0.024), corresponding to about 12% of the fluorescence expected from equivalent concentrations of tryptophan.

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