TABLE 3.

Oxygenation of AA by R120A- and R120Q-containing huPGHS-2 variants at high enzyme to AA ratios

[1-¹⁴C]AA (1 μm) was incubated with the indicated concentration of the huPGHS-2 variant form at 37 °C for 8 min; the reactions were stopped, and the radioactive products and unreacted AA were separated by radio-HPLC and quantified as described under “Experimental Procedures.” The results show the percentage of the original [1-¹⁴C]AA remaining. Values are averages ± S.D. from three reactions. ND means not determined.

Reaction components	Unreacted [1-14C]arachidonic acid remaining after 8 min (% of starting radioactivity)
	Native/Native huPGHS-2	Y385F/Native huPGHS-2	Y385F-R120Q/Native huPGHS-2	Y385F-R120A/Native huPGHS-2	Y385F/R120Q	Y385F/R120A	R120Q/R120Q	R120A/R120A
1 μm AA, 0.1 μm enzyme	4.0 ± 0.7	4.3 ± 1.2	4.7 ± 0.8	7.0 ± 0.9	2.7 ± 0.7	23 ± 4.2	2.4 ± 1.7	ND
1 μm AA, 1 μm enzyme	12 ± 3.0a	8.9 ± 0.8a	9.9 ± 2.4a	1.2 ± 0.4a	30 ± 3.1a	46 ± 3.3a	29 ± 0.6a	1.8 ± 1.2
1 μm AA, 1 μm enzyme, 5 μm PA	3.5 ± 1.9b	4.2 ± 0.5b,c	6.3 ± 0.7b,d	ND	9.9 ± 1.4b,d	28 ± 2.7b,d	20 ± 1.7b,d	ND
1 μm AA, 1 μm enzyme, 60 μm unlabeled AA added after 4 min	2.4 ± 0.5b	2.5 ± 0.5b	1.7 ± 1.3b	ND	ND	2.0 ± 0.6b	3.5 ± 0.2b	ND

^a Significant differences from values with 0.1 μm enzyme were determined using a Student's t test (p < 0.05).

^b Significant differences from values with 1 μm enzyme were determined using a Student's t test (p < 0.05).

^c 10 μm PA instead of 5 μm PA was used.

^d 25 μm PA instead of 5 μm PA was used.