Biosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction.

TitleBiosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction.
Publication TypeJournal Article
Year of Publication1987
AuthorsDavis, JT, Moore, RN, Imperiali, B, Pratt, AJ, Kobayashi, K, Masamune, S, Sinskey, AJ, Walsh, CT, Fukui, T, Tomita, K
JournalJ Biol Chem
Date Published1987 Jan 05
KeywordsAcetyl Coenzyme A, Acetyl-CoA C-Acetyltransferase, Acetyltransferases, Acyl Coenzyme A, Amino Acid Sequence, Chemical Phenomena, Chemistry, Diethyl Pyrocarbonate, Iodoacetamide, Isoelectric Point, Kinetics, Macromolecular Substances, Molecular Weight, Protons, Substrate Specificity, Zoogloea

The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with [2-3H]acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.

Alternate JournalJ Biol Chem
Citation Key215
PubMed ID2878927
Grant List5 RO1 GM33039 / GM / NIGMS NIH HHS / United States