TABLE 3

Summary of CES activity for 4-nitrophenyl esters

Values listed are the average ± S.E. When the kinetic constants K_m and V_max were determined for a substrate and enzyme combination, the intrinsic clearance (CL_int) is V_max/K_m; otherwise, the initial clearance (CL_ini) was calculated. Units for K_m/K_s are micromolar concentration, for V_max are micromoles of product per minute per milligram of protein, and for CL_int/CL_max/CL_ini are microliters per minute per milligram of protein, respectively.

	Kinetic Constants	hCES1	cCES1	dCES1	hCES2^{^a}	cCES2	dCES2^{^b}	hCES3
4NPA	K_m	75.8 ± 6.9	244 ± 10	19.2 ± 1.9	74.4 ± 5.8	186 ± 8	600 ± 70	N.D.
	V_max	40.7 ± 1.8	30.5 ± 0.5	40.6 ± 11.2	44.8 ± 2.1	100 ± 13	5.71 ± 0.64	N.D.
	CL_int	552 ± 79	125 ± 5	2232 ± 744	606 ± 36	539 ± 64	9.54 ± 0.24	N.D.
4NPP	K_m	148 ± 15	101 ± 23	20.0 ± 2.4	200 ± 23	137 ± 2	176 ± 27	N.D.
	V_max	56.1 ± 4.4	38.7 ± 3.5	82.2 ± 10.8	149.4 ± 28.2	164.4 ± 15	16 ± 1.7	N.D.
	CL_int	392 ± 38	448 ± 61	4320 ± 570	858 ± 210	1200 ± 132	93.6 ± 6.6	N.D.
4NPB	K_m	105 ± 18	37.7 ± 2.3	22.5 ± 3.2	97.4 ± 11.1	90.0 ± 14.8	50.3 ± 7.6	81.7 ± 8.1
	V_max	81.6 ± 3.6	20.6 ± 1.5	121.8 ± 12.6	202.8 ± 10.8	511.8 ± 16.8	13.8 ± 0.4	0.690 ± 0.084
	CL_int	834 ± 114	551 ± 38	7500 ± 2100	2172 ± 288	6060 ± 1140	295 ± 47	9.00 ± 1.86
4NPV	K_m	142 ± 22	12.8 ± 1.5	9.65 ± 1.95	67.3 ± 9.3	24.9 ± 3.0	13.5 ± 0.4	N.D.
	V_max	52.1 ± 5	8.04 ± 0.48	41.2 ± 8.1	103.8 ± 13.2	277.2 ± 42.6	12.7 ± 0.2	N.D.
	CL_int	400 ± 77	642 ± 54	4356 ± 420	1554 ± 36	11100 ± 360	936 ± 18	N.D.
4NPDMA^{^c}	K_m	32.5 ± 6.4	10.2 ± 1.7	23.9 ± 2.1	28.9 ± 8.1	13.6 ± 1.5	15.4 ± 1.2	N.D.
	V_max	8.76 ± 1.44	11.7 ± 2.2	80.4 ± 2.4	20.8 ± 5.6	35.2 ± 5.9	2.89 ± 0.1	N.D.
	CL_int	312 ± 84	1140 ± 48	3660 ± 390	888 ± 228	2574 ± 270	192 ± 22	N.D.
	CL_ini-2	9.6 ± 1.98	N.A.	N.A.	38.9 ± 10	70.2 ± 10.8	4.08 ± 0.4	N.D.
4NPTMA	K_m/s	17.4 ± 3.1	13.9 ± 1.2	10.7 ± 0.6	13.5 ± 1.8	25.9 ± 8.6	19.0 ± 0.6	N.D.
	V_max	5.08 ± 0.56	4.41 ± 0.36	26.3 ± 7.7	6.84 ± 1.38	9.66 ± 4.02	4.08 ± 0.4	N.D.
	N	N.A.	N.A.	N.A.	N.A.	N.A.	1.49 ± 0.06	N.D.
	CL_int/max	357 ± 97	320 ± 13	2388 ± 570	511 ± 67	357 ± 30	26 ± 2.9	N.D.
4NPGB^{^d}	K_m	84.4 ± 6.7	848 ± 41	702 ± 144	118 ± 21	45.3 ± 12.5	304 ± 37	N.D.
	V_max	0.636 ± 0.03	0.409 ± 0.044	0.612 ± 0.114	0.285 ± 0.023	39.48 ± 9.18	0.696 ± 0.036	N.D.
	CL_int	7.68 ± 0.6	0.481 ± 0.038	0.888 ± 0.084	2.55 ± 0.2	924 ± 234	2.45 ± 0.25	N.D.
6NC^{^e}	CL_ini	0.139 ± 0.009	0.113 ± 0.01	0.124 ± 0.01	0.125 ± 0.014	0.165 ± 0.012	0.143 ± 0.013	N.D.

N.D., not determined; N.A., not applicable; N, Hill coefficient.
↵a hCES2 showed inhibition at concentrations >50 μM for 4-nitrophenyl trimethylacetate.
↵b dCES2 values for 4-nitrophenyl trimethylacetate are K_s and CL_max instead of K_m and CL_int, respectively.
↵c For the enzymes that displayed biphasic kinetics with 4-nitrophenyl dimethylacetate, the CL_ini-2 is also listed.
↵d cCES2 showed an increased delay in hydrolysis as the substrate concentration increased and inhibition at high substrate concentrations.
↵e Kinetic plots did not plateau for 6-nitrocoumarin with any of the enzymes, thus only the CL_ini could be derived.