Steady-state kinetics inhibitors

In the case of reagents with a low content of inhibitors a steady-state polymerization rate may be set up. Steady-state kinetics are also observed... 

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. 

An inhibitor that binds exclusively to the free enzyme (i.e., for which a = °°) is said to be competitive because the binding of the inhibitor and the substrate to the enzyme are mutually exclusive hence these inhibitors compete with the substrate for the pool of free enzyme molecules. Referring back to the relationships between the steady state kinetic constants and the steps in catalysis (Figure 2.8), one would expect inhibitors that conform to this mechanism to affect the apparent value of KM (which relates to formation of the enzyme-substrate complex) and VmJKM, but not the value of Vmax (which relates to the chemical steps subsequent to ES complex formation). The presence of a competitive inhibitor thus influences the steady state velocity equation as described by Equation (3.1) ... 

Table 3.3 Effects of inhibitors of different modalities on the apparent values of steady state kinetic constants and on specific steps in catalysis... 

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

Henderson PJ. 1972. A linear equation that describes the steady-state kinetics of enzymes and subceUular particles interacting with tightly bound inhibitors. Biochem J 127 321. 

Reversible Inhibition One common type of reversible inhibition is called competitive (Fig. 6-15a). A competitive inhibitor competes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme. Many competitive inhibitors are compounds that resemble the substrate and combine with the enzyme to form an El complex, but without leading to catalysis. Even fleeting combinations of this type will reduce the efficiency of the enzyme. By taking into account the molecular geometry of inhibitors that resemble the substrate, we can reach conclusions about which parts of the normal substrate bind to the enzyme. Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation (Eqn 6-9) becomes... 

The steady-state kinetics of a simple single-substrate, single-binding site, single-intermediate-enzyme catalysed reaction in the presence of competitive inhibitor are shown in Scheme A5.5.1. 

Stable Co111 ADP and ATP complexes have been used as competitive inhibitors in a number of enzymic studies and some progress has been made at unravelling the requirements of the active sites. Steady-state kinetic studies show jff,y-[Co(ATP)(NH3)4] to compete with MnATP for (Na+ + K+), Mg2+ and Ca2+ ATPases derived from kidney medulla.574 values for /J,y-[Co(ATP)(NH3)4] are similar to the Km values for MnATP for both the (Na+ + K+) and Mg24- enzymes, and 3,P NMR shows that the Co111 complex acts as a substrate for the Mg2+ and Ca2+ systems. Likewise,... 

Cleland (160), steady-state kinetics of a Theorell-Chance mechanism can generally apply also to a rapid-equilibrium random mechanism with two dead-end complexes. However, in view of the data obtained with site-specific inhibitors this latter mechanism is unlikely in the case of the transhydrogenase (70, 71). The proposed mechanism is also consistent with the observation of Fisher and Kaplan (118) that the breakage of the C-H bonds of the reduced nicotinamide nucleotides is not a rate-limiting step in the mitochondrial transhydrogenase reaction. 

Steady-state kinetics experiments have shown that anions such as S04 , Cl , and HP04 are inhibitors of the flow of electrons from sulfite to cytochrome c but not to O2 (90, 257). In 1971 Cohen and Fridovich proposed (regarding inhibition by sulfate) that the sulfate sensitive step was not the reduction of the enzyme by sulfite, but was rather the egress of electrons from the enzyme to the 1-electron acceptors (257). 

Just as an appreciation of the forces involved is essential to comprehending the binding of an inhibitor to an enzyme, so is an understanding of the kinetic analysis of an enzyme-catalyzed reaction essential to any kinetic evaluation of an inhibitor. In this section we provide a brief introduction to the study of enzyme kinetics, particularly steady-state kinetics. Regardless, the reader is advised to refer to other sources for more in-depth reviews of the kinetic equations and mathematical derivations involved (38,60, 67-71). 

Sometimes steady-state kinetics are insufficient to analyze the mechanism of inactivation for a given inhibitor. For example, irreversible enzyme inhibitors that bind so tightly to the enzyme that their dissociation rate ( ff) is effectively zero also exhibit noncompetitive inhibition patterns. They act by destroying a portion of the enzyme through irreversible binding, thereby lowering the overall enzyme concentration and decreasing Vmax- The apparent Km remains unaffected because irre-... 

Koder RL and Miller A-F, Steady-state kinetic mechanism, stereospecihcity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase, Biochim. Biophys. Acta, 1387, 395, 1998. 

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