TABLE 5.

Oxygenation of AA by S530A-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, and the reactions were stopped, and the radioactive 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	S530A/S530A huPGHS-2	S530A/Native huPGHS-2	Y385F S530A/Native	Y385F/S530A
1 μm AA, 0.1 μm enzyme	4.0 ± 0.7	4.3 ± 1.2	6.8 ± 1.1	3.2 ± 1.6	3.0 ± 0.4	14 ± 1.3
1 μm AA, 0.5 μm enzyme	ND	ND	1.2 ± 0.05	ND	ND	8.5 ± 1.9
1 μm AA, 1 μm enzyme	12 ± 3.0a	8.9 ± 0.8a	4.3 ± 1.5b	6.5 ± 0.6a	5.2 ± 0.9a	9.2 ± 2.8
1 μm AA, 2 μm enzyme	27 ± 3.7a,c	ND	11 ± 1.1b	11 ± 1.7a	11 ± 0.02a	23 ± 5.5b
1 μm AA, 2 μm enzyme, 25 μm PA	3.5 ± 1.9d,e,f	4.2 ± 0.5e,f,h,	6.7 ± 1.6g	2.4 ± 1.9g	13 ± 1.4g	12 ± 3.2g
1 μm AA, 2 μm enzyme, 60 μm unlabeled AA added after 4 min	2.4 ± 0.5d,f	2.5 ± 0.5d,f	1.5 ± 0.6g	1.5 ± 0.5g	3.4 ± 0.07g	7.0 ± 1.0g

^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 0.5 μm enzyme were determined using a Student's t test (p < 0.05).

^c Data are from Ref. 24.

^d 1 μm enzyme instead of 2 μm enzyme were used.

^e 5 μm PA instead of 25 μm PA were used.

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

^g Significant differences from values with 2 μm enzyme were determined using a Student's t test (p < 0.05).

^h 10 μm PA instead of 25 μm PA were used.