ROLE OF TYR201 AND TYR385 IN SUBSTRATE ACTIVATION BY P-HYDROXYBENZOATE HYDROXYLASE FROM PSEUDOMONAS-FLUORESCENS

The crystal structure of the enzyme-substrate complex of p-hydroxybenz oate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, wh ich is in close contact with the side chain of Tyr385. The role of thi s hydrogen bonding network in substrate activation was studied by kine tic and spectral analysis of Tyr --> Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr2 01 --> Phe and Tyr385 --> Phe) with the physiological substrate are co mparable with those of the corresponding mutant proteins of p-hydroxyb enzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Bal lou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzym e Tyr201 --> Phe has a high K(m) for NADPH and produces only 5% of 3,4 -dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the T yr201 --> Phe mutant does not stabilize the phenolate form of 4-hydrox ybenzoate. With enzyme Tyr385 --> Phe, flavin reduction is rate-limiti ng and the turnover rate is only 2% of wild type. Despite rather effic ient hydroxylation, and deviating from the description of the correspo nding P. aeruginosa enzyme, mutant Tyr385 --> Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate anal ogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimu lates the stabilization of the 4a-hydroperoxyflavin intermediate. Unli ke wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3 ,4-dihydroxybenzoate. The affinity of enzyme Tyr201 --> Phe for the di anionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, p robably because of repulsion of the substrate phenolate in a more nonp olar microenvironment. In contrast to data reported for p-hydroxybenzo ate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxy cinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including seco ndary binding of the inhibitor. Enzyme Tyr201 --> Phe does not stabili ze the phenolate form of the inhibitor. In enzyme Tyr385 --> Phe, the phenolic pK(a) of bound 4-hydroxycinnamate is increased with respect t o wild type. It is proposed that Tyr385 --> Phe is involved in substra te activation by facilitating the deprotonation of Tyr201.