TABLE 2.

Effect of amino acid substitution in NylA on Acd hydrolytic and esterolytic activity

The effects of amino acid substitutions at positions 72, 150, 174, 125, and 316 were tested using purified NylA and its mutant enzymes. Enzyme activities for 10 mm Acd, 0.2 mm p-nitrophenylacetate (C2-ester), 0.2 mm p-nitrophenylbutyrate (C4-ester), and 0.2 mm p-nitrophenyloctanoate (C8-ester) were assayed. The specific activity (μmol min⁻¹(units) mg⁻¹) was estimated for each enzyme. The numbers in parentheses indicate the activity relative to the specific activity of wild-type NylA. Acd hydrolytic activity was assayed in duplicate, and the average value was calculated. The deviations were found to be less than 2%.

	Acd hydrolytic activity	Esterase activity
		C2-ester	C4-ester	C8-ester
	units mg−1	units mg−1
Wild type	4.17 (100)	1.91 ± 0.02 (100)	1.78 ± 0.06 (100)	1.48 ± 0.08 (100)
K72A	<0.002	<0.001	<0.001	<0.001
S150A	<0.001	<0.001	<0.001	<0.001
S174A	<0.001	<0.001	<0.001	<0.001
N125A	0.57 (13.7)	1.37 ± 0.01 (71.7)	1.11 ± 0.02 (62.3)	1.05 ± 0.02 (70.9)
C316A	0.73 (17.5)	1.73 ± 0.01 (90.6)	1.92 ± 0.03 (108)	1.49 ± 0.04 (101)
C316S	4.46 (107)	3.10 ± 0.14 (162)	2.53 ± 0.14 (142)	3.33 ± 0.23 (225)
C316G	7.70 (185)	2.38 ± 0.06 (125)	2.29 ± 0.08 (129)	1.83 ± 0.07 (124)
C316D	0.063 (1.51)	0.82 ± 0.03 (42.9)	1.48 ± 0.08 (83.1)	1.01 ± 0.01 (68.2)
C316E	0.040 (0.96)	1.13 ± 0.02 (59.2)	1.50 ± 0.09 (84.3)	0.96 ± 0.06 (64.9)
C316N	0.18 (4.32)	0.83 ± 0.01 (43.5)	1.12 ± 0.06 (62.9)	0.75 ± 0.05 (50.7)
C316K	0.15 (3.60)	0.84 ± 0.05 (44.0)	1.11 ± 0.01 (62.4)	0.82 ± 0.02 (55.4)