On the reaction mechanism of l -lactate oxidase: Quantitative structure-activity analysis of the reaction with para -substituted  l -mandelates

Abstract

The rate constants for reduction of the f lavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1 H-and 2-2 H-forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects ( 1 H͞ 2 H ‫؍‬ 3-7) on the rate constants of f lavin reduction were found, indicating that f lavin reduction is a direct measure of ␣-C-H bond breakage. The rate constants show only a small inf luence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the f lavin N(5) position or a synchronous mechanism in which the ␣-C-H is formally abstracted as a H ؉ while the resulting charge is simultaneously neutralized by another event.L-Lactate oxidase is a newly studied member of the family of FMN-containing enzymes that catalyze the oxidation of ␣-hydroxyacids, comprising glycolate oxidase (EC 1.1.3.1), Llactate oxidase, L-lactate monooxygenase (EC 1.13.12.4), flavocytochrome b 2 (EC 1.1.2.3), long-chain ␣-hydroxyacid oxidase (EC 1.1.3.15), and L-mandelate dehydrogenase. A considerable amount of mechanistic work has been carried out with L-lactate monooxygenase and with flavocytochrome b 2 (for reviews, see refs. 1 and 2), which has been interpreted as supporting a carbanion mechanism, in which the substrate ␣-proton is abstracted by an active site base and the electrons from the resulting substrate carbanion are transferred to the flavin. The crystal structures of two of the family members, glycolate oxidase (3) and flavocytochrome b 2 (4), have been solved and show an arrangement of conserved protein residues surrounding the FMN prosthetic group and substrate that are consistent with such a carbanion mechanism. In these structures, the substrate is positioned on the si-face of the flavin with the carboxylate in ionic interaction with arginine residues and stabilized by H-bond interaction with a tyrosine residue (5). A conserved histidine residue is located so that the ␣-carbon of the substrate would be positioned between the flavin N(5) and the histidine N1. The histidine residue therefore has been envisaged as the active site base responsible for abstracting the proton from the ␣-position of the substrate, with concomitant attack of the highly nucleophilic carbanion on flavin N(5).While the crystal structures of only two of the family members are available so far, the amino acid sequences of all the enzymes are known: glycolate oxidase (6, 7), flavocytochrome b 2 (8), lactate monooxygenase (9), lactate oxidase (10), long chain ␣-hydroxyacid oxidase (11), and mandelate dehydrogenase (12, 13). All enzymes of the group show considerable homology, and all have the strictly conserved set o...

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

“…In each case, it is clear that the pairs of arginine residues are involved in substrate binding and catalysis. Although the results do not offer any real proof, they are consistent with the hypothesis that the two arginine residues serve the complementary roles of assisting in binding of the substrate carboxylate and in neutralizing the negative charge developed in the transition state on removal of the proton from the ␣-carbon position by the active site histidine base (2).…”

“…The kinetic characteristics of the enzyme have been reported by our group, but still the mechanism of the reductive half-reaction by ␣-hydroxyacids is one of central interest. Our previous study of the reactivity of LOX with a series of para-substituted mandelates indicated little development of charge in the transition state of the reductive half reaction (2). This result is compatible with either a hydride mechanism or with a synchronous mechanism in which the ␣-CH-of the substrate is formally abstracted as a proton, whereas the resulting charge is simultaneously neutralized by another event.…”

“…This result is compatible with either a hydride mechanism or with a synchronous mechanism in which the ␣-CH-of the substrate is formally abstracted as a proton, whereas the resulting charge is simultaneously neutralized by another event. We proposed the participation of two arginine residues, Arg-181 and Arg-268, which are located around the FMN and which could be part of the substrate binding site, based on the x-ray crystal structures of glycolate oxidase (3,4) and flavocytochrome b 2 (5,6). These two arginine residues are conserved in all family members, as shown in Fig.…”

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

“…1996;Mizutani et al, 1996). As a consequence, it was recently suggested that the accepted mechanism of the a-hydroxy acidoxidizing enzymes might also need revision (Pollegioni et al, 1997;Yorita et al, 1997). A discussion of the structural differences at the active site between the two enzyme classes, as well as of examples of differing reactivity with some mechanistically relevant substrates and inactivators lies outside the scope of this article.…”

Revisitation of the βCl-Elimination Reaction of d-Amino Acid Oxidase

“…This puzzle remained unanswered for a long time, as the flavin ought not to be involved directly in elimination. Later experiments based on the concept of the linear free energy relationship were not in favor of the carbanion mechanism either (10,11). A way out of the conundrum emerged when the three-dimensional structure of two related DAAOs was identified (12,13); these studies showed unambiguously that there is no functional group at the active center of the enzyme that could act as a base in abstracting the substrate ␣H as H ϩ .…”