STRENDA׳s requirements that the pH, temperature and substrate concentration be reported are therefore critical in isotope Selleck Kinase Inhibitor Library effect studies as each can influence the magnitude and meaning of the measured KIE (Cook and Cleland, 2007, Cornish-Bowden, 2012 and Segal, 1975). Furthermore, the saturation level of the substrate concentration used should also be noted (e.g. relative to its Km value) in steady-state assays or if pre-steady state rates are reported the

portion of prebound substrate should be mentioned. In addition to the general recommendations of STRENDA, proper error analysis is vital when reporting data from isotope effects. This is especially true for secondary, solvent, equilibrium or heavy atom KIEs since the magnitudes of these values are quite small and therefore can be obscured by the experimental errors Osimertinib molecular weight if careful steps are not taken during the measurement. Even for larger primary KIEs, though, a rigorous error analysis must be carried out since biophysical studies on enzymes often involve measurements over a range of conditions and the conclusions drawn from such studies can be dramatically changed by the uncertainty of the experimental values. One of the probes of quantum mechanical nuclear tunneling in enzymatic C–H activation, for example, relies on measurements

of the temperature dependence of the KIE (Kohen et al., 1999, Nagel and Klinman, 2006, Sutcliffe et al., 2006, Sutcliffe and Scrutton, 2002 and Wang et al., 2012). Temperature independent KIEs and the associated isotope effect on Arrhenius preexponential factors Erlotinib (Al/Ah, where l and h are the light and heavy isotopes, respectively) outside the semi-classical limits are taken as evidence for quantum mechanical tunneling of the hydrogen isotope ( Bell, 1980, Nagel and Klinman, 2006, Nagel and Klinman, 2010, Sutcliffe et al., 2006, Sutcliffe and Scrutton, 2002 and Wang et al., 2012). For KIE data, Arrhenius or Eyring plots, or the isotope effects on

their parameters are identical, as all differences in the rate equations drop out of the ratio equation. Yet visual inspection of Arrhenius or Eyring plots, or simple regression to average values, is often insufficient to determine whether the Al/Ah value is within or outside semiclassical limits (i.e., can be explained without invoking nuclear tunneling). Experimental errors have to always be introduced with even the most sensitive experimental methodologies, to enable assessment of whether the data can be explained by a certain theoretical model or not. Similarly, comparison of KIEs calculated by computer based simulation and experimental data requires both a clear indication of certainty in the calculated values, their distribution (e.g., PES vs. PMF calculations) and the statistical confidence or deviation range of the experimental data from their average value.