Table 1.

Catalytic activity of HbHNL variants, SABP2, and PFE toward cleavage of hydroxynitrile and hydrolysis of p-nitrophenyl acetate

Variant	Hydroxyni trile lyasea	Esterase (p-nitrophenyl acetate)b
	Specific Activity (mU/mg)	Specific Ac- tivity (mU/mg)	kcat (min−1)	Km (mM)	kcat/Km( M−1 min−1)	Fold Esterase Increasec
wild type	30000±100	0.88±0.2	0.11±0.02	1.0±0.07	110	1.0
K236G	npd	npd	npd	npd	npd	npd
T11G	240±30	1.3±0.05	0.09±0.005	0.20±0.04	450	4.1
E79H	1300±39	1.4±0.01	0.08±0.004	0.19±0.04	410	3.7
K236M	36±13	8.4±0.03	0.74±0.02	0.58±0.05	1,270	11.5
T11G K236G	300±14	2.7±0.2	0.13±0.004	0.15±0.02	860	7.8
T11G K236M	≤7	7.3±0.12	0.60±0.06	0.55±0.14	1,080	9.8
E79H K236M	46±10	5.8±0.07	0.32±0.02	0.21±0.04	1,540	14
K236G E79H	≤7	8.0±1.0	0.37±0.01	0.16±0.02	2,260	20.5
T11G E79H	540±40	14.±0.2	0.58±0.02	0.14±0.02	4,160	38
T11G K236M E79H	65±1	14.±0.6	0.59±0.02	0.14±0.02	4,200	38
T11G K236G E79H (TM)	145±30	35.±1.4	1.62±0.05	0.16±0.02	10,100	92
SABP2	≤7	780.±30	120.±6	1.4±0.1	86,000	nae
PFE	nae	19500	1460	0.45	32,400,000	nae

^aRelease of benzaldehyde (ε_{280 nm} = 1380 M⁻¹ cm⁻¹) from mandelonitrile (5 mM) at pH 5.0 measured at 280 nm. Error limits are the standard deviation of three measurements.

^bRelease of p-nitrophenoxide (ε_{404 nm} = 11,580 M⁻¹ cm⁻¹ at pH 7.2) from p-nitrophenyl acetate (0.3 mM) at pH 7.2 measured at 404 nm. Steady state kinetics were measured by varying the p-nitrophenyl acetate concentration from 0.025 mM to 2 mM and fitting the data (5 to 10 data points) to the Michaelis-Menten equation. Errors limits are standard deviations.

^cSpecificity constant (k_cat/K_m) relative to wt.

^dnp = no protein; protein was expressed, but only as insoluble aggregates.

^ena = not applicable.