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Frontier orbital study on the 4âhydroxybenzoateâ3âhydroxylaseâdependent activity with benzoate derivatives
- VERVOORT, Jacques, RIETJENS, Ivonne M. C. M., BERKEL, Willem J. H., VEEGER, Cees
- European journal of biochemistry 1992 v.206 no.2 pp. 479-484
- active sites, catalytic activity, hydroxyl radicals, mutants, pH, phenol
- Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4âhydroxybenzoateâ3âhydroxylase (EC 220.127.116.11). A clear correlation between In kcat for the conversion of a series of 4âhydroxylated 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)âperoxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25Â°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 wildâtype 4âhydroxybenzoateâ3âhydroxylase, its mutants as well as of various other flavinâdependent aromatic hydroxylases, such as for example 3âhydroxybenzoateâ4âhydroxylase (EC 18.104.22.168), 3âhydroxybenzoateâ6âhydroxylase (EC 22.214.171.124) and phenol hydroxylase (EC 126.96.36.199).