Frontier orbital study on the 4‐hydroxybenzoate‐3‐hydroxylase‐dependent activity with benzoate derivatives

Abstract

Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4-hydroxybenzoate-3-hydroxylase (EC 1.14.13.2). A clear correlation between In k_cat for the conversion of a series of 4-hydroxylated substrates and their E(HOMO) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)-peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25°C. Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild-type 4-hydroxybenzoate-3-hydroxylase, its mutants as well as of various other flavin-dependent aromatic hydroxylases, such as for example 3-hydroxybenzoate-4-hydroxylase (EC 1.14.13.23), 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7).