Studies on the Mechanisms of Action of Phenylalanine Hydroxylase and Its Protein Stimulator I. ENZYME CONCENTRATION DEPENDENCE OF THE SPECIFIC ACTIVITY OF PHENYLALANIKE HYDROXYLASE DUE TO A NONENZYMATIC STEP

The specific activity of phenylalanine hydroxylase from rat liver decreases markedly with increasing enzyme concentration when assayed at pH 8.0 with the naturally occurring tetrahydrobiopterin as cofactor. The decrease in specific activity is due to a sharp increase in the apparent K, for tetrahydrobiopterin at higher enzyme concentration, whereas V,,, is unchanged. This protein concentration dependence is relieved by the presence of a protein, phenylalanine hydroxylase-stimulating protein, which has been purified from rat liver (HUANG, C. Y., MAX, E. E., AND KAUFMAN, S. (1973) J. Biot. Chem. 248, 4235). The stimulating protein greatly increases the apparent affinity of the hydroxylase for tetrahydrobiopterin, but has no effect on V Ill&X. Because of its ability to stimulate phenylalanine hydroxylase activity, the stimulating protein served as a valuable probe for the mechanism of actionof the hydroxylase. Kinetic and physical studies reported here show that the dependence of specific activity on enzyme concentration is not due to the existence of different molecular weight forms of phenylalanine hydroxylase having different affinities for tetrahydrobiopterin. Neither is it due to the presence of an inhibitor which is present at a constant ratio to the enzyme. Our data are consistent with the concept that the protein concentration dependence of the specific activity of phenylalanine hydroxylase arises from the peculiar kinetic process of the enzymatic reaction. It is proposed that an intermediate is released from the enzyme during catalysis, which can recombine with the enzyme or break down nonenzymatically to product (or products). The slower nonenzymatic step (relative to the rate of recombination) imposes a limit on the over-all reaction rate, thereby resulting in an apparent enzyme concentration dependence. The proposed role of the stimulating protein is the catalysis of the nonenzymatic step. Our observations also suggest that the intermediate is either a precursor of the quinonoid dihydro form of biopterin or a complex involving biopterin, oxygen, and phenylalanine. XII2 + TPNH + II+ + XHI + TPN+ (DPNH) (DPN+) (2)