Factors Affecting the Steady State Kinetic Constants

1 Factors Affecting the Steady State Kinetic Constants 

We can now relate the kinetic constants kCM, Ku, and kcJKM to specific portions of the enzyme reaction mechanism. From our discussions above we have seen that the term kCM relates to the reaction step of ES conversion to ES. Hence experimental perturbations (e.g., changes in solution conditions, changes in substrate identity, mutations of the enzyme, and the presence of a specific inhibitor) that exclusively affect kCM are exerting their effect on catalysis at the ES to ES transition step. The term KM relates mainly to the dissociation reaction of the encounter complex ES returning to E + S. Conversely, the reciprocal of Ku (1IKU) relates to the association step of E and S to form ES. Inhibitors and other perturbations that affect the 

For our purposes the most important factor that can impact the individual steady state kinetic constants is the presence of an inhibitor. We will see in Chapter 3 how specific modes of inhibitor interactions with target enzymes can be diagnosed by the effects that the inhibitors have on the three steady state kinetic constants. 

Other factors that can impact these constants relate to reaction solution conditions. We have already discussed how temperature can affect the value of kCM and kcJKM according to the Arrhenius equation (vide supra). Because enzymes are composed of proteins, and proteins undergo thermal denaturation, there are limits on the range of temperature over which enzymes are stable and therefore conform to Arrhenius-like behavior. The practical aspects of the dependence of reaction velocity on temperature are discussed briefly in Chapter 4, and in greater detail in Copeland (2000). 

The effects of solution pH on enzyme activity can be particularly informative in defining steps in catalysis that are most affected by interactions with inhibitors. Ionization of different groups on the enzyme can be critical in substrate binding (i.e., 

---