title: The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera.
abstract: Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912.
authors.forename: V E
authors.initials: VE
authors.lastname: Anderson
authors.forename: B J
authors.initials: BJ
authors.lastname: Bahnson
authors.forename: I D
authors.initials: ID
authors.lastname: Wlassics
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism*; D015045:Zoogloea / Q000201:enzymology*
chemical_list: D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C059703:acetyldithio-coenzyme A; D013869:Thiolester Hydrolases