10.1074\/jbc.m009177200<\/a><\/p>\nAuthors:<\/h4>\n
Lee-Ho Wang, Ah\u2010Lim Tsai, Pei-Yung Hsu<\/p>\n
Abstract:<\/h4>\n
Thromboxane synthase (TXAS) is a \u00ab\u00a0non-classical\u00a0\u00bb cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. 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 ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 x 10(4) M(-1) s(-1) and 1.5 x 10(5) M(-1) s(-1) for imidazole and clotrimazole, respectively, followed by a slow conformational 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 hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min(-1) and that the k(cat)\/K(m) for PGH2 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-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, 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 parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2-2.0 x 10(7) M(-1) s(-1); the rate of catalytic conversion of PGH2 to TXA2 or MDA was at least 15,000 s(-1) and the lower limit of the rates for products release was 4,000-6,000 s(-1). Given that the cellular PGH2 concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with \u00ab\u00a0classical\u00a0\u00bb P450 enzymes.<\/p>\n","protected":false},"excerpt":{"rendered":"
DOI: 10.1074\/jbc.m009177200 Authors: Lee-Ho Wang, Ah\u2010Lim Tsai, Pei-Yung Hsu Abstract: Thromboxane synthase (TXAS) is a \u00ab\u00a0non-classical\u00a0\u00bb cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[],"class_list":["post-646","post","type-post","status-publish","format-standard","hentry","category-freeze-quench"],"_links":{"self":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/646","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=646"}],"version-history":[{"count":0,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/646\/revisions"}],"wp:attachment":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=646"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=646"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=646"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}