title: Coenzyme A dithioesters: synthesis, characterization and reaction with citrate synthase and acetyl-CoA:choline O-acetyltransferase.
abstract: Acyl dithioesters of CoA have been synthesized by transesterification. The alpha-hydrogens have a spectrally determined pKa of 12.5 +/- 0.14. The hydroxide catalyzed enolization rate is estimated to be 600 M-1.s-1. The absorbance of the dithioester, lambda max = 306 nm, can be used to monitor both the condensation and transesterification reactions that use CoA-Ac as a substrate. For citrate synthase at pH 7.4 Vmax = (4.0 +/- 0.4).10(-4) s-1 and Km = 53 +/- 7.5 microM, which are 2.10(-6) and 3.3-times the Vmax and Km values observed for CoAS-Ac, while for Ac-CoA: choline O-acetyltransferase (EC 2.3.1.6) at pH 7.0 Vmax = (1.1 +/- 0.2).10(-2) mumol.s-1.(mg protein)-1 and Km = 83 +/- 33 microM, which are 0.077 and 10-times the values observed with CoAS-Ac, respectively. The CoA dithioesters are stable at low pH, but hydrolyze with a second-order rate constant of 8.2.10(-2) M-1.s-1 at pH 11.4. The spectral properties of these dithioesters should allow these analogs to be used as probes of the structure of enzyme bound intermediates.
authors.affiliation: Department of Chemistry, Brown University, Providence, RI 02912.
authors.forename: I D
authors.initials: ID
authors.lastname: Wlassics
authors.forename: C
authors.initials: C
authors.lastname: Stille
authors.forename: V E
authors.initials: VE
authors.lastname: Anderson
mesh_terms: D000214:Acyl Coenzyme A / Q000138:chemical synthesis*; D002795:Choline O-Acetyltransferase; D002950:Citrate (si)-Synthase; D004795:Enzyme Stability; D004952:Esters; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007652:Oxo-Acid-Lyases; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D004952:Esters; D002795:Choline O-Acetyltransferase; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases