Abstract
The importance of cysteine residues in the cyclooxygenase activity of prostaglandin endoperoxide synthase (PGHS) was investigated using cysteine-specific reagents and site-directed mutagenesis. N-(7-Dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM), a hydrophobic maleimide, inactivated both cyclooxygenase and peroxidase activities of apoPGHS in a time-dependent manner but did not affect holoPGHS. Heme titration experiments indicated that modification of apoPGHS with DACM prevented heme binding. Peptide mapping revealed that DACM modified Cys313, Cys512, and Cys540. N-Ethylmaleimide inactivated cyclooxygenase and peroxidase activities of holoPGHS in a time-dependent manner but did not affect apoPGHS. Peptide mapping demonstrated that N-ethylmaleimide reacted primarily with Cys313 in holoPGHS and with Cys540 in apoPGHS. Each of the 3 cysteines was changed to serine by site-directed mutagenesis, and the mutant proteins were expressed in COS-1 cells. The C512S mutant converted arachidonic acid to products to the same extent as wild-type PGHS. In contrast, the C313S and C540S mutants converted arachidonic acid to products to the extent of 10% of wild-type PGHS. These results indicate that Cys313, Cys512, and Cys540 are not essential for cyclooxygenase activity but that alteration of Cys540 or Cys313 dramatically decreases enzyme activity. Both residues are well removed from the cyclooxygenase and peroxidase active sites so our findings reveal that subtle changes, such as substitution of a single oxygen for sulfur atom as far as 30 A from the heme prosthetic group, can significantly alter enzyme activity.
Highlights
Agents and site-directed mutagenesis.N-('ir-Dimethyl- The cyclooxygenase reaction is initiated by the stereospecific amino-4-methyl-3-couarinyl)maleimid(eDACM), a hy- removal of the 13-pro-S hydrogen of arachidonic acid, and the drophobic maleimide, inactivated both cyclooxygenase identity of the oxidizing agent is thseubject of intense scrutiny and peroxidase activities of apoPGHS in a time-depend- [7,8]
Incubationof apoPGHS with a 22-fold excess of DACM at pH 7 resulted in a time-dependent loss of cyclooxygenase and peroxidase activities
ApoPGHS reconstituted with hematinwas not affected by DACM
Summary
Tored for peptides at 214 nm anfdor DACM-modifiedpeptides at 383nm simultaneously with a Hewlett Packard model 1040A diode array de-. Assays were initiated by the addition of hydrogen peroxide to give a containing template for mutagenesis of Cys3I3.A 479-bp EcoRI-XbaI final concentration of 400 p ~ P.eroxidase activity was calculated as fragment from codon 501 to the TGA stop codon 600 plus 182 bp of described previously [29]. Inactiuation of PGHS by Mateimides-Purified apoprotein was diluted to 10 p~ with 100 mM sodium phosphate, pH7.0, containing 0.1% Tween 20 in thepresence or absencoef 10 p~ hematin Inactivation was initiated tbhye addition of 9.8 p l tion and isolation of the mutant M138, the sequences of the entire of either 10mM DACM in acetone or 1m0 ~ NEM in water gtoive a final subcloned fragments of mutated cDNAs were verified by dideoxy seconcentration of 220 p ~ C.orrecting for dilution the final enzyme con- quencing of template preparations. C.) The conversion of arachidonic acid to products was linear with respect t o both protein content and incubation time
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