TABLE 4.

Acetylation of huPGHS-2 variants with aspirin

huPGHS-2 variant	Stoichiometry of aspirin acetylation ([14C]acetyl/dimer)a	FA oxygenase activity remaining after aspirin treatmentb	Eicosanoid productsc
			PGH2-derived	HETEs
		% (FA units/mg)	% total products	% total products
His8-Native/His8-Native		100 (45 units/mg)	91	9
His8-Native/His8-Native + ASA	0.94 ± 0.06 (n = 3)	47 (21 units/mg)	62 (53d)	38 (47d)
His8-Y385F/FLAG-Native		100 (42 units/mg)	82	18
His8-Y385F/FLAG-Native + ASA	0.80 ± 0.11 (n = 4) (p = 0.12)	38 (16 units/mg)	18	82
His8-Y385F/His8-Y385F			0	0
His8-Y385F/His8-Y385F + ASA	0.11 ± 0.09e (n = 2)	ND (0/0)	ND	ND
His8-R120A/His8-R120A		100 (26 units/mg)	86	14
His8-R120A/His8-R120A + ASA	0.16 ± 0.04e (n = 2)	91 (24 units/mg)	84	16
His8-Y385F-R120A/FLAG-Native			92	8
His8-Y385F-R120A/FLAG-Native + ASA	ND	ND	19	81
S530A/S530A		100 (16 units/mg)	79	21
S530A/S530A + ASA	0.06 ± 0.01e (n = 2)	100 (16 units/mg)	87	13
His8-Y385F/FLAG-S530A		100 (12 units/mg)	80	20
His8-Y385F/FLAG-S530A + ASA	−0.04 ± 0.05e (n = 2)	110 (13 units/mg))	89	11
His8-Y385F-S530A/FLAG-Native		100 (37 units/mg)	80	20
His8-Y385F-S530A/FLAG-Native + ASA	0.74 ± 0.03e (n = 2) (p = 0.036)	48 (18 units/mg)	20	80
S530A/Native		100 (30 units/mg)	97	3
S530A/Native + ASA	0.47 ± 0.04e (n = 4) (p = 0.006)	74 (22 units/mg)	54	46

^a Incorporation of [¹⁴C]acetyl group from [¹⁴C]acetylsalicylate was determined as described under “Experimental Procedures.”

^b Oxygenase activity was determined using a standard COX assay with 100 μm AA, and the value for O₂ consumption was corrected for FA turnover as described in the legend to Fig. 1.

^c Eicosanoid products formed from 100 μm [1-¹⁴C]AA were analyzed by radio-HPLC as described under “Experimental Procedures.”

^d Data are from Ref. 23.

^e Significant difference from [¹⁴C]acetylsalicylate-treated His₈-Native/His₈-Native huPGHS-2 was determined by Student's t test (p < 0.05).