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. 2004 Feb;13(2):466–475. doi: 10.1110/ps.03416804

Table 4.

ATP-dependent kinetic parameters for wild-type and mutant CPS

Glutamine-dependent ATPase Inline graphic-dependent ATPase CP-dependent ATP synthesis
Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) Km (mM) kcat (s−1) kcat/Km (mM−1 s−1)
WT 0.029 ± 0.002 4.04 ± 0.1 137.5 0.005 ± 3.10 • 10−4 0.09 ± 2.9 • 10−3 17.9 0.006 ± 0.001 0.36 ± 0.01 65.3
A144Q 0.285 ± 0.056 3.94 ± 0.51 13.9 0.034 ± 0.007 0.10 ± 0.01 2.9 0.005 ± 3.9 • 10−5 0.28 ± 8.0 • 10−4 56.4
D207A 0.015 ± 0.002 1.51 ± 0.11 100.9 0.006 ± 0.001 0.11 ± 0.01 18.9 0.006 ± 0.001 0.26 ± 0.02 44.6
S209A 0.031 ± 0.004 2.43 ± 0.13 78.0 0.016 ± 0.001 0.35 ± 0.01 22.7 0.011 ± 0.001 0.59 ± 0.01 51.4
ADS 0.571 ± 0.083 0.66 ± 0.04 1.1 0.319 ± 0.012 0.33 ± 0.01 1.0 0.008 ± 0.001 0.28 ± 0.01 36.3
D207N 0.020 ± 0.018 1.65 ± 0.02 84.2 0.009 ± 0.003 0.06 ± 0.01 7.2 0.004 ± 0.002 0.14 ± 0.01 36.6
A144Q/D207A 0.154 ± 0.001 1.70 ± 0.08 11.0 0.021 ± 0.002 0.12 ± 0.01 5.8 0.011 ± 4.0 • 10−4 0.37 ± 0.04 33.9
I211S 0.072 ± 0.009 4.77 ± 0.26 66.4 0.020 ± 0.002 0.29 ± 0.01 14.4 0.011 ± 0.002 0.48 ± 0.02 44.0
P690Q 0.383 ± 0.061 9.01 ± 0.79 23.5 0.009 ± 0.002 0.09 ± 0.01 10.4 0.039 ± 0.005 0.28 ± 0.01 7.1
D753A 0.064 ± 0.010 1.71 ± 0.13 26.8 0.007 ± 4.0 • 10−4 0.14 ± 2.9 • 10−3 19.4 0.002 ± 2.0 • 10−4 0.07 ± 2.4 • 10−3 41.2
F755A 0.135 ± 0.011 5.18 ± 0.17 38.3 0.004 ± 0.001 0.07 ± 2.4 • 10−3 14.8 0.031 ± 0.004 0.28 ± 0.01 9.0
PDF 0.199 ± 0.015 2.90 ± 0.11 14.6 0.022 ± 0.005 0.34 ± 0.03 15.6 0.027 ± 0.004 0.06 ± 3.5 • 10−3 2.3
D753N 0.121 ± 0.026 5.60 ± 0.59 46.2 0.008 ± 0.001 0.13 ± 0.01 16.9 0.005 ± 3.0 • 10−4 0.15 ± 2.4 • 10−3 31.1

ATPase reactions contained 50 mM HEPES, 100 mM KCl, 20 mM MgSO4, 40 mM NaHCO3, 10 mM ornithine, 1 mM DTT, 10 mM phosphoenolpyruvate, 0.2 mM NADH, 18 u/mL pyruvate kinase, and 24 u/mL lactate dehydrogenase in the presence or absence of 10 mM glutamine. ATP synthesis reactions included 50 mM HEPES, 100 mM KCl, 20 mM MgSO4, 10 mM CP, 10 mM ornithine, 1 mM NADP, 20 mM glucose, 10 u/mL hexokinase, and 5 u/mL glucose-6-phosphate dehydrogenase. All reactions were performed with 5 or 50 μg CPS in 500 μL final volume at 25°C, final pH 7.6. Standard error of the kinetic parameters was determined from nonlinear regression curve fitting (GraFit, version 5.01) and was within ±6.1%.