NMR Analysis of Covalent Intermediates in Thiamin Diphosphate Enzymes

Abstract

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…Using a combined acid quench/ 1 H NMR-method (Tittmann et al, 2003), the relative distribution of ThDP and of the acid-stable intermediates HBz-ThDP and benzoin-ThDP can be assessed. At first, the enzymes were washed three times with 50 mM KH 2 PO 4 /K 2 HPO 4 buffer at 4°C to remove excess ThDP.…”

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

“…Rapid mixing and NMR analysis experiments were carried out and analyzed by the method of Tittmann et al (9). A hexahistidinefused enzyme (15 mg͞ml enzyme) was reconstituted with 250 M FAD, 10 mM Mg 2ϩ , and an equimolar (active site concentration) amount of ThDP, in 0.1 M KPi buffer (pH 7.6).…”

“…After separation of the denatured protein by centrifugation, the distribution of ThDP intermediates in the protein-free solution was analyzed by 1 H NMR spectroscopy (9). The integrated areas of the C6Ј-H signals were used to determine the relative amounts of the covalent intermediates in the solution, whereas the amount of ThDP itself was determined from the area of the C2-H peak because its C6Ј-H signal was obscured by signals because of FAD (see, for example, Fig.…”

“…For each of these mutant proteins, the distribution of the intermediates bound to ThDP during turnover was determined by the rapid mixing͞quench NMR method and compared with the behavior of the WT enzyme described in ref. 9. Reactions were carried out in the presence of 100 mM pyruvate, or a mixture of 50 mM pyruvate and 50 mM 2-ketobutyrate, and the enzymatic reaction mixture was quenched with trichloroacetic acid͞DCl in D 2 O.…”

“…Unless the rate constants of parallel steps with pyruvate and 2-ketobutyrate as the second substrate are coincidentally identical, it further implies that the rate-determining step occurs before binding of the second substrate. Tittmann et al (9) recently used an acid-quench͞ 1 H NMR technique to assess the distribution of ThDP-containing intermediates in wild-type (WT) AHAS II at the steady state and showed that the slowest step(s) by far in the reaction is the formation of the first covalent adduct, lactyl-ThDP. It thus is not surprising that the turnover rate for WT AHAS II is unaffected by the competition between second substrates.…”