. Author manuscript; available in PMC: 2020 Nov 12.

Published in final edited form as: Biochemistry. 2019 Nov 4;58(45):4480–4493. doi: 10.1021/acs.biochem.9b00653

Table 3.

Kinetics of Fe(II)- and Zn(II)-S39E and wild-type HDAC8^a

HDAC8	k_cat/K_M (M⁻¹s⁻¹)	K_M (μM)	k_cat (s⁻¹)	k_cat/K_M Ratio (WT/S39E)	k_off (min⁻¹)	k_off Ratio (S39E/WT)
Fe(II)-S39E	440 ± 60	170 ± 30	0.077 ± 0.005	6 ± 2	0.48 ± 0.05	9 ± 1
Fe(II)-WT	2800 ± 700	90 ± 30	0.25 ± 0.02	6 ± 2	0.055 ± 0.005	9 ± 1
Zn(II)-S39E	26 ± 3	> 400^b	> 0.05^b	37 ± 6	0.57 ± 0.07	14 ± 3
Zn(II)-WT	950 ± 96	1200 ± 300	1.1 ± 0.3	37 ± 6	0.040 ± 0.006	14 ± 3

Values for k_cat/K_M, K_M, and k_cat were obtained by fitting the Michaelis-Menten equation (Equation 1) to the dependence of the initial rates of deacetylation on the substrate concentration catalyzed by Zn(II)- and Fe(II)-constituted S39E and wild-type HDAC8 as measured using the FdL assay. Enzyme concentration was 0.5–1 μM and substrate concentration was varied from 10–1000 μM. Values for k_off were determined by fitting a single exponential (Equation 2) to the time-dependent decrease in activity upon incubation with 1 mM EDTA. Standard errors for k_cat/K_M, K_M, k_cat, and k_off values were calculated using GraphPad Prism analysis. Error for k_cat/K_M and k_off ratios were calculated using the propagation of uncertainty equation.⁴⁰

Little curvature was observed in the dependence of activity on substrate concentration so that K_M and k_cat values are poorly defined by this data set. The necessity for excessively high enzyme and substrate concentrations preclude the accurate determination of these parameters, individually.