TY - JOUR A1 - Johnson, Kenneth A. T1 - New standards for collecting and fitting steady state kinetic data JF - Beilstein Journal of Organic Chemistry PY - 2019/// VL - 15 SP - 16 EP - 29 SN - 1860-5397 DO - 10.3762/bjoc.15.2 PB - Beilstein-Institut JA - Beilstein J. Org. Chem. UR - https://doi.org/10.3762/bjoc.15.2 KW - computer simulation KW - data fitting KW - enzyme catalysis KW - induced-fit KW - Michaelis constant KW - specificity constant N2 - The Michaelis–Menten equation is usually expressed in terms of kcat and Km values: v = kcat[S]/(Km + [S]). However, it is impossible to interpret Km in the absence of additional information, while the ratio of kcat/Km provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, kcat/Km provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis–Menten equation in terms of kcat and kcat/Km values: v = kSP[S]/(1 + kSP[S]/kcat), where the specificity constant, kSP = kcat/Km. In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of kcat/Km can be derived directly using the modified form of the Michaelis–Menten equation rather than calculated from the ratio of kcat and Km values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive kcat and kcat/Km is illustrated by considering the role of conformational changes in enzyme specificity where kcat and kcat/Km can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret Km, which is best understood as the ratio of kcat divided by kcat/Km. 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