Abstract
Thromboxane synthase is a ferrihemoprotein which undergoes mechanism-based inactivation during catalysis. This "suicide" process may be an important factor for limiting thromboxane A2 biosynthesis in cells. Although the kinetics have been characterized for purified enzyme and platelets, the chemical basis for inactivation has remained unclear. Protein modification or alteration of the heme prosthetic group is each compatible with the irreversible nature of suicide inactivation of thromboxane synthase. We have investigated these two possibilities using enzyme purified to homogeneity. Our data show that the Soret absorbance spectrum of thromboxane synthase is unaltered by additions of prostaglandin endoperoxide H2 which cause enzymatic inactivation. Using a coupled cyclooxygenase/thromboxane synthase system and polyacrylamide gel electrophoresis we have demonstrated that the enzyme retains radiolabel under nondenaturing gel conditions. Label incorporation is reduced by the competitive thromboxane synthase inhibitor U63557, an agent that also protects the enzyme from inactivation. Under denaturing conditions the radiolabel localizes with the released heme prosthetic group. In addition, interaction of the heme prosthetic group with cyanide was prevented by inactivating the enzyme with prostaglandin H2. In similar experiments, the lipid hydroperoxide 15(S)-hydroperoxyeicosatetraenoic acid inactivated thromboxane synthase with concurrent bleaching of the Soret spectrum. Labeling studies with a coupled soybean lipoxygenase/thromboxane synthase system indicate that, in this case, the apoenzyme is modified. These results suggest that the mechanism of thromboxane synthase inactivation during thromboxane A2 biosynthesis involves a tight, nondestructive association of substrate or product with the prosthetic heme group. Inactivation by hydroperoxides, however, appears to result from apoenzyme modification. These reactions may have important implications for cellular physiology and pathophysiology of thrombosis.
Highlights
Thromboxane synthase is a ferrihemoprotein which typified by inhibition of cyclooxygenase in platelets,is characundergoes mechanism-basedinactivation during catal-terizeedxtensively
Al- processes restraining TxA2 formation; “suicide” inthough the kinetics havebeen characterized for puri- activation of Txsynthase, a mechanism-basedeventthat fied enzyme and platelets, thechemical basis for inactivation has remained unclear
Proteminodification or alteration of the heme prosthetic group is each compatible with the irreversible natureof suicide inactivation of thromboxane synthase.We have investigated these twopossibilities using enzymepurified tohomogeneity
Summary
Synthase activity emerged in the flow-through fraction. After ultraf’iltration, this sample was diluted with 0.01 M phosphate buffer, pH. ’Tx synthase in samples collected at 0.5-min intervals was located by incubatingportions (0.1ml, 0.1-0.3 mg of protein/ml) from each sample with 10 p~ PGH2 for 1 min and monitoring TxB, formation cant reductionof the heme absorbance of the enzyme at 418420 nm (Fig. 1, upper panel). Incorporation of radiolabel into apoenzyme during retic analysis and autofluorography indicated that this radiolabeled material did not all correspond to [I4C]arachidonic acid or its metabolites: a portion migrated with heme, suggesting an association (Fig. 4, lanes 3 and 4 and lanes 7 and 8).Addition of heme to inactive enzyme and incubation at transformation of [’“Clarachidonic acid by the cyclooxygenase/Tx synthase coupled system: denaturing gel electrophoresis. Consistent with the involvementof the heme prosthetic group in“suicide” inactivation.Cyanide reduced the Soret absorbance and altered the shapeof the absorbance spectra of Tx synthase; half-maximal loss of absorbance at 418-420 nm occurred with 7mM NaCN (Fig. 5 , upper panel).U63557a. I69 cox protected the enzyme from alteration; changes in the absorption spectrum were only evident a t cyanide concentrations approaching 80 mM (Fig. 5 , middlepanel).Likewise, “suicide”-
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