TABLE 2.
Kinetic constants of AcXbh30A in the hydrolysis of pNP-X2.
| Kinetic constant | AcXbh30A pNP-X2 kinetic valuesa , b |
|---|---|
| K M | 116.4 ± 3.23 μM |
| V max | 183.1 ± 1.13 U mg−1 |
| k cat | 152.7 s−1 |
| K cat /K M | 1,311.6 s−1 mM−1 |
| K i (X 2 ) c | 2.4 mM |
Standard assay consisted of 100 μl reactions containing 30 mM sodium acetate pH 4.0, 0.02 mg/ml BSA, variable concentrations of pNP-X2 in the range of 30 μM–4 mM, and 5 ng of AcXbh30A-CD GEP enzyme which was incubated at 65 C for 10 min. The reaction was stopped and developed by addition of 300 μl of 200 mM sodium carbonate and measured at 405 nm. Baseline corrected data were converted to concentration of pNP using its reported mM extinction coefficient (ε = 18.4).
For substrate concentration–dependent kinetics, three separate assays were performed. A least-squares fit of the non-linear Michaelis–Menten enzyme kinetics model in GraphPad Prism 8 was used to obtain kinetic values of the three curves as a group. The error is reported as the standard error of parameters, and the standard error of fit was 2.532 U/mg.
Product inhibition was studied by inclusion of X2 in the standard pNP-X2 assay at concentrations of 0.5, 1, 2, and 4 mM. No X2, 0.5 mM X2, and 1.0 mM X2 datasets were performed at least twice, while the datasets for 2 and 4 mM X2 were only performed a single time. A robust fit of the non-linear competitive enzyme inhibition model in GraphPad Prism 8 was used to obtain the inhibition constant (K i).