. Author manuscript; available in PMC: 2013 Jan 10.

Published in final edited form as: Biochemistry. 2011 Dec 29;51(1):243–256. doi: 10.1021/bi2015508

Table 3.

Steady-state kinetic parameters of Pnc1 mutants. Assays were performed as described under Experimental Procedures. All reactions were performed at pH 7.5.

Pnc1 mutant	k_cat (s⁻¹)^a	K_m (µM)^a	k_cat/K_m (M⁻¹s⁻¹)	¹⁵N KIE	¹³C KIE
wild type	0.69 ± 0.04	9.6 ± 2.1	(7.2 ± 1.2) × 10⁴	1.0122 ± 0.0002	1.0125 ± 0.0004
D8A	0.0009 ± 0.0001	NA^b	NA^b	ND^c	ND^c
D8N	0.0006 ± 0.0001	NA^b	NA^b	ND^c	ND^c
D8E	0.0070 ± 0.0001	NA^b	NA^b	1.0218 ± 0.0015	1.0258 ± 0.0012
D51A	0.020 ± 0.005	< 2.4	> 8.3 × 10³	1.0166 ± 0.0018	1.0075 ± 0.0015
D51N	0.064 ± 0.012	< 1.8	> 3.5 × 10⁴	1.0047 ± 0.0006	1.0058 ± 0.0006
H53A	0.014 ± 0.002	6.5 ± 3.6	(2.2 ± 0.9) × 10³	ND^c	ND^c
H94A	0.020 ± 0.002	< 2.7	> 7.4 × 10³	ND^c	ND^c
K122A	0.044 ± 0.023	< 6.5	> 6.7 × 10³	ND^c	ND^c
K122R	0.0009 ± 0.0002	NA^b	NA^b	1.0127 ± 0.0003	1.0172 ± 0.0001
C167A	< 0.0005	NA^b	NA^b	ND^c	ND^c

k_cat and K_m values were determined from fitting the data to the Michaelis-Menten equation using Kaleidagraph (Synergy Software, Reading, PA). Errors represent the error of the fit to the data.

Not available. Measured rates were too slow for accurate K_m values to be determined.

Not determined.