title: Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.
abstract: The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Johns Hopkins University, Baltimore, Maryland 21205-2179, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Harris
authors.forename: M W
authors.initials: MW
authors.lastname: Washabaugh
mesh_terms: D000079:Acetaldehyde / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D011522:Protons; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D012440:Saccharomyces / Q000201:enzymology*; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D011522:Protons; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde; D013835:Thiamine Pyrophosphate