Alternative equations and “pseudo-statistical” approaches that enhance the precision of initial rates for the determination of kinetic parameters

A burning concern among researchers studying enzyme kinetics has been ways of improving the accuracy of initial rates ( v ) with much greater precision. The goal of this study was to establish a formal (mathematical) way of achieving more accurate v values in enzyme assay. By adopting Bernfeld method of assay, the v values generated and other values in the literature were explored with two main objectives:1) to derive equations for a correctional calculation of initial rates and for the calculation of Michaelis-Menten (MM) parameters and 2) to evaluate the derived equations. The results of study showed that the maximum velocity ( V max ) and the MM constant ( K M ) obtained from a robust nonlinear regression (RNR) using v values in the literature were respectively between 21 and 26-fold and 6.56 to 61.2-fold greater than values generated from other method such as double reciprocal plot (drp), reciprocal variant of direct linear plot (RVDLP), and new method (z-model) derived based on MM equation; the V max and K M values (for alpha-amylase) calculated based on the RVDLP using the uncorrected v values were respectively 1.26- and 1.264-fold greater than the values using corrected v values; based on the z method, the V max and K M values using the uncorrected v values were 179.8- and 9.35-fold greater than the values using their corrected counterparts. In conclusion, the erroneous initial rates can be corrected using new equations before fitting RNR, RVDLP, drp, and any other equation to the corrected v values for the determination of MM parameters. Graphical Abstract figure The implication of the result in b (i) is that the V max and K M should either be a negative value or infinite value according to the equations unlike b (ii).