title: Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.
abstract: Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
authors: Kenneth M
authors: KM
authors: Roberts
authors: Jorge Alex
authors: JA
authors: Pavon
authors: Paul F
authors: PF
authors: Fitzpatrick
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D006900:Hydroxylation; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D011622:Pterins; D014443:Tyrosine; D010649:Phenylalanine; C017523:6-methyltetrahydropterin; D010651:Phenylalanine Hydroxylase