. Author manuscript; available in PMC: 2011 Dec 15.

Published in final edited form as: Biochemistry. 2010 Nov 15;49(49):10421–10439. doi: 10.1021/bi1012518

Table 3.

Nicotinamidase and ¹⁸O exchange activity of SpNic and mutants

SPNic	Catalytic rate^a (k_obs, s⁻¹)	¹⁸O Exchange rate^d (k_obs, s⁻¹)
wt	3.4^b	0.34
R97A	3.2^b	0.27
K103A	0.0053^c	0.0076
C136A	<10⁻⁶^c	<10⁻⁶
C136S	<10⁻⁶^c	ND^c

The observed rate of nicotinamidase catalysis of SPNic and mutants measured using 200 µM NAM at 37 °C.

The rates were determined by GDH plate reader assay.

The rates were determined by HPLC assay. The full experimental is available in the Methods section.

The observed ¹⁸O exchange into nicotinic acid catalyzed by SpNic and mutants as measured by the initial rate method and mass spectrometry (Figure 4) in the presence of 1 mM nicotinic acid and ¹⁸O H₂O at 37 °C. Enzyme concentrations were 100 nM SpNic wt and R97A and 5 µM for K103A or C136A mutants. The measured rate was corrected by the mole fraction of ¹⁸O in the reaction as described by equation 4 in the Methods. Full experimental is in Methods section. ^cN/D: not determined.