Kinetics and mechanism of benzoylformate decarboxylase using carbon-13 and solvent deuterium isotope effects on benzoylformate and benzoylformate analogs

Abstract

Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide. The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF). The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive). Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed. Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation. Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors. These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).

Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I

“…Considering the available information about reactions involving TPP (12,17,20) plus the data obtained in the present study, we propose for the reaction catalyzed by benzaldehyde lyase the mechanism schematized in Fig. 4 (13).…”

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense

“…The molecular mass of the holoenzyme was estimated by size-exclusion chromatography on a Superdex G200 column, equilibrated with 10 mM MES, 2.5 mM MgSO 4 , 0.5 mM ThDP, 150 mM KCl, pH 6.5 (flow rate of 1 ml min Ϫ1 ). The molecular mass was estimated by comparing the elution volume (at least three repetitions) with those of known standard proteins (molecular mass in kDa is indicated in parentheses): RNase A (13.7), chymotrypsinogen A (25), ovalbumin (43), albumin (67), aldolase (158), catalase (232), ferritin (440), and thyroglobulin (669). Blue dextran 2000 was used to determine the void volume of the column.…”

“…The decarboxylation activity was measured using a coupled photometric assay with alcohol dehydrogenase as an auxiliary enzyme (43). A typical reaction mixture (pH 6.5) contained 10 mM MES, 2.5 mM MgSO 4 , 0.1 mM ThDP, 0.35 mM NADH, and 0.125 U alcohol dehydrogenase from horse liver.…”

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

“…A reaction mechanism for the decarboxylation has been proposed for PDC (Kluger, 1987) and BFD (Weiss et al, 1988;Reynolds et al, 1988;Iding et al, 2000). According to these investigations, the reaction is catalyzed directly at the cofactor ThDP with an enaminecarbanion as intermediate.…”

“…Besides acetaldehyde, BFD converts aromatic and heteroaromatic substrates as acceptors to produce enantiopure (R)-benzoin and derivatives, but in contrast to BAL with lower reaction rates (Iding et al, 2000). BFD basically follows the same mechanism as BAL (Figure 2-7) with the only difference that no cleavage of the formed (R)-2-hydroxy ketones could be observed so far (Reynolds et al, 1988;Iding et al, 2000;Weiss et al, 1988). Тhe benzoin-forming activity of BFD was enhanced by site-directed mutagenesis of histidine 281 to alanine, yielding more space in the active site for accommodating larger acceptor aldehydes (Demir et al, 1999).…”