"Suicide" inactivation of thromboxane A2 synthase. Characteristics of mechanism-based inactivation with isolated enzyme and intact platelets.
"Suicide" inactivation occurs during catalysis by thromboxane synthase. Loss of enzymatic activity, accompanying thromboxane B2 formation, was proportional to the substrate concentration. Inactivation was directly related to product formation: for several different experimental protocols 50% loss of thromboxane synthase activity corresponded with formation of 454 +/- 79 ng of thromboxane B2/mg protein. The time course of inactivation was pseudo-first-order and obeyed saturation kinetics. Inactivation (KI) and first-order rate constants (ki) were 18 microM and 0.18 s-1 for prostaglandin H2. Prostaglandin H1, a poor substrate for turnover, was also a site-directed inactivator with KI = 28 microM and ki = 0.09 s-1. Competitive inhibitors, typified by U63557a and U46619, preserved the enzyme activity by slowing the rate of inactivation from 0.18 to 0.05 s-1. Loss of the hemoprotein Soret absorbance did not correlate quantitatively or temporally with the loss of thromboxane synthase activity. A similar, irreversible inactivation accompanied thromboxane formation by intact platelets. Loss of activity was proportional to substrate concentration and catalytic activity. For a pool of 25 separate donors, thromboxane synthase activity declined exponentially as a function of thromboxane B2 formation: 50% loss of activity corresponded to 23 ng of thromboxane B2/10(7) platelets. The data conform to criteria for a specific, mechanism-based process in which thromboxane synthase participates in two parallel reactions, one leading to thromboxane formation and the other to suicide inactivation. The specific, rather than indiscriminate, nature of the process, and its occurrence in intact platelets may have implications for the cell biology of thrombosis. Depletion of thromboxane synthase activity may be a factor in the choice and effectiveness of antithrombotic agents.
Jones
D A
DA
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Fitzpatrick
F A
FA
D001782:Blood Donors; D001792:Blood Platelets / Q000201:enzymology*; D002384:Catalysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D044262:Prostaglandin H2; D011453:Prostaglandins / Q000494:pharmacology; D011463:Prostaglandins H / Q000494:pharmacology; D013930:Thromboxane-A Synthase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification
D011450:Prostaglandin Endoperoxides, Synthetic; D011453:Prostaglandins; D011463:Prostaglandins H; D044262:Prostaglandin H2; D013930:Thromboxane-A Synthase