Substrate binding is the rate-limiting step in thromboxane synthase catalysis

Thromboxane synthase (TXAS) is "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxyge n, it uses prostaglandin H-2 (PGrH(2)) to catalyze either an isomerization reaction to form thromboxane A(2) (TXA(2)) or a fragmentation reaction to f orm 12-L-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA(2):heptadecatrienoic acid:MDA), We report here kinet ics of TXAS with heme ligands in binding study and with PGH, in enzymatic s tudy. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105-130 s(-1); 2) binding of cyanide, a carbon- based ligand, is a one-step process with k(on) of 2.4 M-1 s(-1) and k(off) of 0.112 s(-1); and 3) both imidazole and clotrimazole (nitrogen-based liga nds) bind TXAS in a two-step process; an initial binding to the heme-iron w ith on-rate constants of 8.4 x 10(4) M-1 s(-1) and 1.5 x 10(5) M-1 s(-1) fo r imidazole and clotrimazole, respectively, followed by a slow conformation al change with off-rate constants of 8.8 s(-1) and 0.53 s(-1), respectively . The results of our binding study indicate that the TXAS active site is hy drophobic and spacious. In addition, steady-state kinetic study revealed th at; TXAS consumed PGH(2) at a rate of 3,800 min(-1) and that the k(cat)/K-m for PGH(2) consumption was 3 x 10(6) M-1 s(-1). Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-f low experiments revealed marginal absorbance changes upon mixing TXAS with PGH(2), indicating minimal accumulation of any heme-derived intermediates, Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state, Further kinetic analysis using a combination of rapid- mixing chemical quench and computer simulation showed that the kinetic para meters of TXAS-catalyzed reaction are: PGH(2) bound TXAS at a rate of 1.2-2 .0 x 10(7) M-1 s(-1): the rate of catalytic conversion of PGH(2) to TXA(2) or MDA was at least 15,000 s(-1) and the lower limit of the rates for produ cts release was 4,000-6,000 s(-1). Given that the cellular PGH(2) concentra tion is quite low, we concluded that under physiological conditions, the su bstrate-binding step is the rate-limiting step of the TXAS-catalyzed reacti on, in sharp contrast with "classical" P450 enzymes.