FLAVIN MOTION IN P-HYDROXYBENZOATE HYDROXYLASE - SUBSTRATE AND EFFECTOR SPECIFICITY OF THE TYR222-]ALA MUTANT

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts wi th the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mu tant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Ty r222 is possibly involved in flavin motion, which may facilitate the e xchange of substrate and product during catalysis. To elucidate the fu nction of Tyr222 in more detail. in the present study the substrate an d effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenz oate hydroxylase, was investigated. Replacement of Tyr222 by Ala impai rs the binding of the physiological substrate 4-hydroxybenzoate and th e substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydr oxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydrox ybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H. A., M attevi, A., Oblomova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T. & van Berkel, W. J. H (1994) Biochemistry 33, 10161-10170] sugg est that this is due to motion of the flavin ring out of the active si te, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hy droxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dih ydroxybenzoate/mol NADPH oxidized. This indicates that reduction of th e Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towa rds the productive 'in' position. [A222]p-Hydroxy benzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate . The regioselectivity of hydroxylation suggests that [A222]p-hydroxyb enzoate hydroxylase binds the fluorinated substrate in the same orient ation as wild-type. Spectral studies suggest that wildtype and [A222]p -hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the ph enolate form with the flavin ring preferring the 'out' conformation. D espite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydr oxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the prote in-substrate interactions at the rr-side of the flavin ring.