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

Abstract

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 (Vmax) and the MM constant (KM) 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 Vmax and KM 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 Vmax and KM 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 O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/524223v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@2d6dcborg.highwire.dtl.DTLVardef@833f08org.highwire.dtl.DTLVardef@9f4648org.highwire.dtl.DTLVardef@bf2e9f_HPS_FORMAT_FIGEXP M_FIG C_FIG The implication of the result in b (i) is that the Vmax and KM should either be a negative value or infinite value according to the equations unlike b (ii).