. Author manuscript; available in PMC: 2017 Sep 5.

Published in final edited form as: Biochemistry. 2016 Sep 19;55(39):5554–5565. doi: 10.1021/acs.biochem.6b00383

Table 1.

Kinetic Constants for EcCTPS at 37°C^a

limiting substrate	condition^a	S_0.5 (μM)	Hill coeff, n_H
UTP		59 ± 15	1.4 ± 0.3 (23)^b
	150 μM ATP	200 ± 40	1.8 ± 0.1 (6)
ATP		130 ± 10	1.7 ± 0.2 (18)^c
	60 μM UTP	310 ± 20	1.9 ± 0.4 (5)
glutamine^d		360 ± 20^e	1.1 ± 0.1 (11)

effector	condition^a	IC₅₀/EC₅₀ (μM)

CTP		IC₅₀	370 ± 60^f (6)
	50 μM UTP 100 nM EcCTPS	IC₅₀	160 ± 10^g (4)
GTP^h		EC₅₀	38 ± 3 (4)
		IC₅₀	540 ± 60

Unless noted, substrates were present at saturation with optimal GTP. ([UTP] = 600 μM, [ATP] = 1500 μM, [GTP] = 200 μM, [glutamine] = 10 mM). S_0.5 and n_H values were determined by fitting data to the Hill function. IC₅₀ values were estimated by linear extrapolation of data points straddling 50% inhibition values (see Materials and Methods). The number of experiments used for each determination is given in parentheses.

The S_0.5 and n_H values did not significantly vary from 10 to 2000 nM enzyme (at 100 nM, S_0.5 = 57 ± 8, n_H = 1.3 ± 0.2, from seven experiments).

The S_0.5 and n_H values did not significantly vary from 50 to 400 nM enzyme (at 100 nM, S_0.5 = 126 ± 13, n_H = 1.7 ± 0.3, from six experiments).

These values were determined at 100 nM enzyme and include four values determined in the presence of 500 μM NADH, which had no discernible effect.

Lineweaver–Burke analysis gave 411 ± 44 μM as the K_m value. Bearne and Iyengar reported a K_m of 320 μM using HPLC.⁵⁸

The limiting slopes of the biphasic Hill plots were −1.22 ± 0.3 and −4.8 ± 1.9, at the low and high concentration ranges, respectively.

The limiting slopes of the biphasic Hill plots were −0.6 ± 0.2 and −1.9 ± 0.3, at the low and high concentration ranges, respectively.

GTP EC₅₀ and IC₅₀ values were estimated by linear extrapolation of data points straddling 50% activation and inhibition values, respectively. Nonlinear Hill plots yielded limiting slopes of 1.8 ± 0.2 and −4.6 ± 0.3, at the low and high concentration ranges, respectively. Fitting the data from individual experiments to a two-site model,¹⁰ where n_act = 1, gave the following averaged parameters: k_act = 10.0 ± 0.6 s⁻¹, K_A = 57 ± 7 μM, K_i = 308 ± 24 μM, and n_inh = 5.0 ± 0.4. Previously reported values were, respectively, 10.3, 23, 190, and 3.8.¹⁰