Substrate recognition by "password" in p-hydroxybenzoate hydroxylase

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science 266, 110-114; Schreuder, H. A., Mat tevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic stu dies detected the movement of the flavin from the buried conformation to th e exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reducti on in wild-type PHBH and the His72Asn mutant indicates that the deprotonati on of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonat ion to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before commi tting to the catalytic cycle.