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B7. Meaning of Kinetic Constants

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  • It is important to get a "gut-level" understanding of the significance of the rate constants. Here they are:

    • Km: The Michaelis constant with units of molarity (M), is operationally defined as the substrate concentration at which the initial velocity is half of Vmax. It is equal to the dissociation constant of E and S only in if E, S and ES are in rapid equilibrium. It can be thought of as an "effective" Kd in other cases.
    • kcat: The catalytic rate constants, with units of s-1 is often called the turnover number. It is a measure of how many bound substrate molecules turnover or form product in 1 second. This is evident from equation v0 = kcat[ES]
    • kcat/Km: Under condition when [S] << Km, the Michaelis-Menten equation becomes v0 = (kcat/KM)[E0][S]. This really describes a biomolecular rate constant, with units of M-1s-1, for conversion of free substrate to product. Some enzyme have kcat/Km values around 108, indicating that they are diffusion controlled. That implies that the reaction is essentially done as soon as the enzyme and substrate collide. The constant kcat/Km is also referred to as the specificity constant in that it describes how well an enzyme can differentiate between two different competing substrates. (We will show this mathematically in the next chapter.)
    Table: Km and kcat values for various enzymes
    Km values
    enzyme substrate Km (mM)
    catalase H2O2 25
    hexokinase (brain) ATP 0.4
    D-Glucose 0.05
    D-Fructose 1.5
    carbonic anhydrase HCO3- 9
    chymotrypsin glycyltyrosinylglycine 108
    N-benzoyltyrosinamide 2.5
    b-galactosidase D-lactose 4.0
    threonine dehydratase L-Thr 5.0
    kcat values
    enzyme substrate kcat (s-1)
    catalase H2O2 40,000,000
    carbonic anhydrase HCO3- 400,000
    acetylcholinesterase acetylcholine 140,000
    b-lactamase benzylpenicillin 2,000
    fumarase fumarate 800
    RecA protein (ATPase) ATP 0.4
    Table: diffusion controlled enzymes
    Enzymes with kcat/Km values close to diffusion controlled (108 - 109 M-1s-1)
    enzyme substrate kcat (s-1) Km (M) kcat/Km (M-1s-1)
    acetylcholinesterase acetylcholine 1.4 x 104 9 x 10-5 1.6 x 108
    carbonic anhydrase CO2 1 x 106 1.2 x 10-2 8.3 x 107
    HCO3- 4 x 105 2.6 x 10-2 1.5 x 107
    catalase H2O2 4 x 107 1.1 4 x 107
    crotonase crotonyl-CoA 5.7 x 103 2 x 10-5 2.8 x 108
    fumarase fumarate 8 x 102 5 x 10-6 1.6 x 108
    malate 9 x 102 2.5 x 10-5 3.6 x 107
    triose phosphate isomeraser glyceraldehyde-3-P 4.3 x 103 4.7 x 10-4 2.4 x 108
    b-lactamase benzylpenicillin 2.0 x 103 2 x 10-4 1 x 108