Where Initial Rates are Directly Proportional to Substrate Concentrations with Application in Molar-Mass Determination, Zero-Order Specificity Constant is Inappropriate

"High-ranking scientists" use the initial rate (v_i) equation regardless of the conditions under which it can be applied. This approach incorrectly suggests that v_i is directly proportional to the substrate concentration, [S₀], where the proportionality constant is V_max (which is an inappropriate maximum velocity) divided by Michaelis-Menten constant, K_M instead of enzyme-substrate complex dissociation, K_d. The main objectives are threefold: 1) demonstrating that v_i is not equal to V_max [S₀]/K_M, 2) establishing that K_d is directly proportional to the enzyme concentration ([E₀]), and 3) illustrating that the standard quasi-steady-state assumptions (sQSSA) and reverse QSSA (rQSSA) have a limited range of validity. The study utilized both experimental and theoretical approaches, following the Bernfeld method for enzyme assay. A K_M-like value was determined to be 2.569 g/L, higher than the putative K_d of 2.482 g/L. Other K_M-like values recorded were 2.396 and 2.407 g/L, corresponding to K_d values of 2.288 and 2.299 g/L, respectively. The molar mass of insoluble potato starch ranged from 62.296 to 65.616 exp. (+6) g/mol.  When the [S₀] is much lower than K_M, the derived equations contradict the assumption that v_i equals V_max/K_M. Additionally, K_d was found to be directly proportional to[E₀]. Furthermore, the molar mass of starch could be determined using the given equations. Graphical and mathematical evidence showed that the sQSSA and rQSSA domains are valid only within certain limits. The equation for determining the second-order rate constant based on rQSSA validity does not apply to sQSSA. The KM-like value may exceed the putative Kd value.