Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases

摘要

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 ± 1s~(-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 μM, 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.