TABLE 1

Michaelis-Menten kinetics parameters of naturally occurring CKM mutations Recombinantly expressed and purified CKM mutants were incubated with creatine and variable ATP conc. along with a pyruvate kinase/lactate dehydrogenase system. ATP dephosphorylation was measured by changes in NADH conc. monitored by absorbance at 340 nm that were the result of the coupled lactate dehydrogenase reaction. The assay was performed with n = 3, and the data were globally fitted to the Michaelis-Menten equation. Kinetic parameters are presented as fitted values or calculated values based on fitted parameters with standard error. A linear regression produced the best fit of the R132C data, thus only a fitted kcat/Km value was able to be obtained and separate kcat and Km values are reported as N/A (not applicable).

Mutantkcat (s−1)Km (mM)kcat/Km (M−1s−1)
WT29.15 ± 1.501.271 ± 0.28922,930 ± 5340
T35I26.96 ± 1.621.870 ± 0.46014,410 ± 3650
R43Q21.93 ± 0.992.090 ± 0.37510,490 ± 1940
T52N22.39 ± 1.241.047 ± 0.26621,390 ± 5560
I92M20.19 ± 1.391.406 ± 0.41914,290 ± 4370
H97Y11.68 ± 1.238.513 ± 2.5771371 ± 439
R130H13.41 ± 1.8416.49 ± 5.34813.1 ± 286.2
R132CN/AN/A210.5 ± 12.4
F169L19.13 ± 1.731.220 ± 0.49215,680 ± 6480
Y173C14.37 ± 0.793.009 ± 0.6154775 ± 1011
W211R16.74 ± 3.4830.35 ± 12.28551.6 ± 250.9
F250S24.53 ± 1.630.9823 ± 0.304224,960 ± 7910
N274Y22.52 ± 1.191.237 ± 0.29118,200 ± 4380
V280L25.09 ± 1.391.423 ± 0.34017,630 ± 4330
N286I15.20 ± 2.8822.33 ± 9.09680.7 ± 305.7
L317M40.98 ± 2.850.7334 ± 0.253455,880 ± 19,700