INVESTIGATION OF THE ROLE OF CYSTEINES IN CATALYSIS BY PROSTAGLANDIN ENDOPEROXIDE SYNTHASE

The importance of cysteine residues in the cyclooxygenase activity of prostaglandin endoperoxide synthase (PGHS) was investigated using cyst eine-specific reagents and site-directed mutagenesis. N-(7-Dimethylami no-4-methyl-3-coumarinyl)maleimide (DACM), a hydrophobic maleimide, in activated both cyclooxygenase and peroxidase activities of apoPGHS in a time-dependent manner but did not affect holoPGHS. Heme titration ex periments indicated that modification of apoPGHS with DACM prevented h eme binding. Peptide mapping revealed that DACM modified Cys(313), Cys (512), and Cys(540). N-Ethylmaleimide inactivated cyclooxygenase and p eroxidase activities of holoPGHS in a time-dependent manner but did no t affect apoPGHS. Peptide mapping demonstrated that N-ethylmaleimide r eacted primarily with Cys(313) in holoPGHS and with Cys(540) in apoPGH S. Each of the 3 cysteines was changed to serine by site-directed muta genesis, and the mutant proteins were expressed in COS-1 cells. The C5 12S mutant converted arachidonic acid to products to the same extent a s wildtype PGHS. In contrast, the C313S and C540S mutants converted ar achidonic acid to products to the extent of 10% of wild-type PGHS. The se results indicate that Cys(313), Cys(512), and Cys(540) are not esse ntial for cyclooxygenase activity but that alteration of Cys(540) Or C ys(313) dramatically decreases enzyme activity. Both residues are well removed from the cyclooxygenase and peroxidase active sites so our fi ndings reveal that subtle changes, such as substitution of a single ox ygen for sulfur atom as far as 30 Angstrom from the heme prosthetic gr oup, can significantly alter enzyme activity.