A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck

Bioresources and Bioprocessing

Table 3 Kinetic data of wild type and the I19T/R34G/R262H variant on ATP and UTP when different concentrations of CTP are present

Enzyme	Sub	Vmax (μM/min)			K_M (mM)			k_cat (s⁻¹)
Enzyme	CTP (mM)	0	0.2	0.4	0	0.2	0.4	0	0.2	0.4
WT	ATP^a	131.0 ± 2.0	26.9 ± 2.2	23.0 ± 3.0	1.08 ± 0.05	0.44 ± 0.02	–^c	1.31 ± 0.01	0.26 ± 0.03	0.23 ± 0.01
variant	ATP^a	314.8 ± 6.2	208.6 ± 1.8	177.4 ± 2.4	0.77 ± 0.06	0.61 ± 0.01	0.48 ± 0.02	3.14 ± 0.05	2.08 ± 0.07	1.77 ± 0.02
WT	UTP^b	80.2 ± 3.5	31.8 ± 1.3	25.5 ± 2.3	0.53 ± 0.01	0.17 ± 0.03	0.13 ± 0.05	0.80 ± 0.01	0.32 ± 0.02	0.25 ± 0.02
variant	UTP^b	210.9 ± 5.2	133.5 ± 3.2	124.3 ± 4.7	0.79 ± 0.06	0.82 ± 0.01	0.94 ± 0.05	2.10 ± 0.09	1.33 ± 0.02	1.24 ± 0.01

^aATP concentrations range from 0.2 to 5.0 mM and UTP concentrations are fixed at 10 mM
^bUTP concentrations range from 0.2 to 5.0 mM and ATP concentrations are fixed at 10 mM
^cNot detected