Relationship between the substrate activation site and catalytic site of phenylalanine hydroxylase.

Abstract

Rat liver phenylalanine hydroxylase when activated with phenylalanine becomes catalytically competent. In the experiments presented, the stoichiometry of binding of phenylalanine to activated rat liver phenylalanine hydroxylase was measured. It was found 1) that there is 1 phenylalanine activation site/subunit of catalytically active enzyme, implying 4 activation sites/active phenylalanine hydroxylase molecule (tetramer); 2) that tryptophan (which is also a substrate for this enzyme) will not bind at the activation site, demonstrating that the activation site and catalytic site of the enzyme are not identical; additional indirect evidence suggests that the activation site and catalytic site of the enzyme do not physically overlap at all; and 3) that lysolecithin (which can also activate this enzyme) and phenylalanine do not appear to directly compete for the same activation site. An amino acid analysis of rat liver phenylalanine hydroxylase is presented; also, an addition to the purification procedure for phenylalanine hydroxylase (Shiman, R., Gray, D. W., and Pater, A. (1979) J. Biol. Chem. 254, 11300-11306) is described that allows one to obtain electrophoretically homogeneous enzyme of apparently 100% purity.