The catalytic mechanism of ascorbate peroxidase : probing the effects of changes in enzyme and substrate structure

Abstract

Recombinant pea cytosolic ascorbate peroxidase (rAPX) has been isolated and the mechanistic properties have been investigated. Rate constants for formation of compound I with H2O2 and other organic peroxides were measured. The data indicate that the structure and size of peroxide dictate the rate of compound I formation. Rate constants for reduction of compound I and compound II were measured with L-ascorbic acid and other derivatised forms of ascorbate. Reduction of compound II showed evidence for formation of an enzyme-substrate complex. The rate constants for compound II reduction, by the various ascorbate-based substrates, were controlled by the thermodynamic driving force of the reaction.;Variants H42A and H42E were constructed to investigate the catalytic role of His42 in rAPX catalysis. The observed pseudo-first-order rate constant for the reaction between the His42 variants and hydrogen peroxide saturates at high peroxide concentration. The data are consistent with a two-step mechanism involving the formation of an APX-H2O2 intermediate whose conversion to compound I is rate-limiting. pH-Dependence studies on compound I formation reveal His42 as the key ionizable residue. Rapid photodiode array spectrophotometry revealed the presence of a transient intermediate for H42A, with a spectrum consistent with a ferric-hydroperoxy complex. The rate of formation of compound I and peroxidase activity of H42E were significantly greater than H42A, however, addition of exogenous imidazoles partially rescues both the rate of compound I formation and peroxidase activity for H42A