2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions

Journal Article · Mon Nov 13 23:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00449a018· OSTI ID:6855538

Gruys, K J; Datta, A; Frey, P A ^[1]

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1. Univ. of Wisconsin, Madison (USA)

Rate constants for the hydrolysis of acetyl-TPP were measured pH values of 2.5 and 7.5 and plotted as log k{sub obs} versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pK{sub a} of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is promoted below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP have facilitated the isolation and identification of (1-{sup 14}C)acetyl-TPP from acid-quenched enymatic reaction mixtures at steady states. (1-{sup 14}C)Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E{sub 1}). The pH-rate profile for hydrolysis of (1-{sup 14}C)-acetyl-TPP, isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the {sup 14}C-labeled enzymic species.

• OSTI ID: 6855538
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:23; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AZINES
AZOLES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
CARBON 14 COMPOUNDS
CHEMICAL REACTIONS
CHEMISTRY
DECOMPOSITION
ENZYMES
HEMIACETAL DEHYDROGENASES
HETEROCYCLIC COMPOUNDS
HYDROLYSIS
KINETICS
LABELLED COMPOUNDS
LYSIS
MAGNETIC RESONANCE
NUCLEAR MAGNETIC RESONANCE
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PH VALUE
PHOSPHORUS COMPOUNDS
PYRIMIDINES
PYROPHOSPHATES
REACTION KINETICS
RESONANCE
SOLVOLYSIS
THIAZOLES