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 flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3-7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of alpha-C-H bond breakage. The rate constants show only a small influence 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 flavin N(5) position or a synchronous mechanism in which the alpha-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Figure 1

Stopped flow traces of the reduction of A95G and wild-type lactate oxidase with l-mandelate. Equal volumes of argon-equilibrated enzyme solution (12 μM final concentration) were mixed with argon-equilibrated l-mandelate solutions of different concentration, and reduction of the flavin monitored at 456 nm. Conditions were pH 7.0 at 25°C. The traces with wild-type enzyme, curves from right to left, were obtained with l-mandelate concentrations of 1, 1.5, 2.5, 5, 10, and 25 mM. The traces with A95G enzyme, curves right to left, were obtained with l-mandelate concentrations of 1, 2, 5, and 10 mM.

Figure 2

Determination of the reduction rate constants (k_red) and dissociation constant (K_d) for reaction of wild-type lactate oxidase with α-¹H-l-mandelate and α-²H-dl-mandelate. Reaction traces such as those of Fig. 1 are strictly single exponential in character, yielding at each concentration a pseudo-first order rate constant, k_obs. The reciprocal of k_obs is here plotted vs. reciprocal of the l-mandelate concentration, to yield values of 1/k_red from the y-intercept and K_d from the slope/intercept (24). The data shown are for α-¹H-l-mandelate (•) and α-²H-dl-mandelate (▴), with the concentration of the latter expressed as the l-form.

Figure 3

Hammett plots of the reduction rate constants of A95G (circles) and wild-type (triangles) lactate oxidase by α-¹H- (solid symbols) or α-²H- (open symbols) forms of p-substituted l-mandelates. The derivatives used were dl-hydroxy-, l-methoxy-, l-methyl-, l- and dl- H-, l-fluoro-, l-chloro-, l-trifluoromethyl- and l-nitro-mandelates. The σ-parameters of p-substituted functional groups were taken from ref. .

Figure 4

Multiple regression analyses of the data of Fig. 2 and Table 1, omitting the data points for hydroxy- and nitro-mandelates. Symbols are as in Fig. 3. See text for details, and Table 2 for numerical details of the regression analyses.

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