Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases.
Phenylalanine hydroxylase (PAH) is a pterin-dependent non-heme metalloenzyme that catalyzes the oxidation of phenylalanine to tyrosine, which is the rate-limiting step in the catabolism of Phe. Chromobacterium violaceum phenylalanine hydroxylase (cPAH) has been prepared and its steady-state mechanism has been investigated. The enzyme requires iron for maximal activity. Initial rate measurements, done in the presence of the 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH(4)) cofactor, yielded an average apparent k(cat) of 36+/-1 s(-1). The apparent K(M) values measured for the substrates DMPH(4), L-Phe, and O(2) are 44+/-7, 59+/-10, and 76+/-7 microM, respectively. Steady-state kinetic analyses using double-reciprocal plots revealed line patterns consistent with a sequential ter-bi mechanism in which L-Phe is the middle substrate in the order of binding. The occurrence of a line intersection on the double-reciprocal plot abscissa when either pterin or O(2) is saturated suggests that, prior to O(2) binding, DMPH(4) and L-Phe are in associative pre-equilibrium with cPAH. Together with an inhibition study using the oxidized cofactor, 7,8-dimethyl-6,7-dihydropterin, it is conclusive that the mechanism is fully ordered, with DMPH(4) binding the active site first, L-Phe second, and O(2) last. This represents the first conclusive steady-state mechanism for a PAH enzyme.
Volner
Alon
A
Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E Young Drive East, Los Angeles, CA 90095-1569, USA.
Zoidakis
Jérôme
J
Abu-Omar
Mahdi M
MM
D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002861:Chromobacterium / Q000201:enzymology*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism; D011622:Pterins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D011621:Pteridines; D011622:Pterins; D011994:Recombinant Proteins; C025596:7,8-dihydropterin; C005063:2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine; D007501:Iron; D010105:Oxygenases; D010651:Phenylalanine Hydroxylase; D010100:Oxygen