Direct Estimate of the Specificity Constant: A Possibility or a Fluke? Pre-steady-state Substrate Concentrations and Enabling Mathematical Equations

Recently, a high-ranking scientist proposed the need for direct estimation of the specificity constant, rather than calculation based on the determination of maximum velocity and the Michaelis–Menten constant ( ). The goal of this work is to develop new formulas for the following: the saturating substrate concentration ( ); the instantaneous initial rate, also called the "burst phase-like" rate; the zero-arbitrary determination of the pre-steady-state (PSS) substrate concentration, which is suitable for PSS assays; the direct estimation of the specificity constant (SC) in a PSS scenario; and its corresponding   («  ). In order to show the proposed equations' applicability and robustness, the paper also aims to quantitatively evaluate them. The research was theoretical as well as experimental. The Bernfeld method of enzyme assay provides evidence for it. For three distinct enzyme concentrations, the SC values from the two most recent approaches range from 2,197.546 to 11,101.74 L/g. min using one way and from 2,185.649 to 13,860.014 L/g min using the other. For all three concentrations, the SC's sub-  values fall between 1,304.368 and 7,943 L/g min. The corresponding   and burst-phase-like initial rate ( ) range from 14.26 to 55.448 μmol/min and 0.171 to 3.752 g/L, respectively. Lower enzyme concentrations result in greater SC values; formulas for directly computing SC under circumstances that validate the reverse and conventional quasi-steady-state approximations could be developed. SC and catalytic efficiency are two quite distinct ideas. Computation cannot be totally avoided.