TABLE 2.

Kinetic parameters of the modified forms of E. coli UMP kinase at two pH values^a

Enzyme	pH 6			pH 8
	KmUMP (mM)	KmATP (mM)	VmaxUMP, ATP (μmol/min/mg of protein)	KmUMP (mM)b	KmATP (mM)	VmaxUMP, ATP (μmol/min/mg of protein)
Wild typec	0.17 ± 0.03	0.048 ± 0.004	105 ± 5	0.043c ± 0.002	0.12c ± 0.018	128 ± 11
Mutants
R62H	1.26 ± 0.21	0.24 ± 0.03	1.5 ± 0.03	0.32 ± 0.04	3.0 ± 0.2	4.4 ± 0.1
D77N	1.78 ± 0.30	0.12 ± 0.02	1.9 ± 0.20	0.47 ± 0.06	0.48 ± 0.12	8.2 ± 0.3
D146N	0.45 ± 0.009	0.03 ± 0.006	0.15 ± 0.01	0.059 ± 0.02	0.10 ± 0.03	0.32 ± 0.03
D159N	0.27 ± 0.06	0.095 ± 0.003	128 ± 13	0.052c ± 0.02	0.29c ± 003	153 ± 5
D168N	0.32 ± 0.06	0.14 ± 0.006	63 ± 4.2	0.06 ± 0.003	0.42 ± 0.01	86.6 ± 19
D174N	3.70 ± 0.36	0.17 ± 0.04	32 ± 3.5	0.31 ± 0.06	0.31 ± 0.06	45 ± 5
D201N	0.86 ± 0.16	0.20 ± 0.04	1.04 ± 0.20	0.22 ± 0.06	0.46 ± 0.09	8.2 ± 0.3

^aThe reaction medium is described in Materials and Methods. The apparent K_m for ATP and UMP was determined at a single, fixed concentration of cosubstrates. The V_max^{UMP, ATP} was obtained by extrapolating the reaction rates for infinite concentrations of ATP and UMP and by assuming that the concentration of one nucleotide substrate does not affect the apparent K_m of the second nucleotide substrate. The mean values and the standard errors of the means are from three separate experiments. 

^bAt pH 8 (or 7.4), where inhibition by excess of UMP occurred, the reaction rates were fitted by the equation v = V_max [UMP]/(K_m^UMP + [UMP] + [UMP]²/K_I). The calculated K_I values were 1.2 mM (wild type), 0.7 mM (D168N), 0.4 mM (D174N), and 0.65 mM (D159N). The other variants of E. coli UMP kinase did not exhibit inhibition by excess of UMP. 

^cFrom reference 15, kinetic measurements being done at pH 7.4.