Specificity constant inhibitors

Two proteinase inhibitors, Inhibitors I and II, accumulate in leaves of tomato plants when attacked by chewing Insects or mechanically wounded. The accumulation of these two antinutrient proteins is apparently a defense response and is initiated by the release of a putative wound hormone called the proteinase Inhibitor inducing factor (PIIF). The direction of flow of PIIF out of wounded leaves is primarily towards the apex and transport occurs maximally about 120 min following wounding. After a single severe wound, the vitro translatable tomato leaf mRNA specific for Inhibitors I and II Increases to a maximum within four hours and remains constant for about five hours when it decreases rapidly to about 50% of the maximum. 

Concise, basic enzyme data is recorded for some 800 enzymes including the Enzyme Commission number, trivial name, systematic name, reaction catalyzed, equilibrium constant, molecular weight, specific activity, specificity, Michaelis constants, inhibitors, light absorption data, and references. Where required, conditions, source, etc. are specified. 

In our earlier study (2) we measured the concentration of ions required to inactivate the enzyme and tried to determine whether the inactivation could be changed by competition with the normal ions, Mg2+, Na+, and K From these studies we assigned the inactivation effects of some cations to actions at specific sites. The cations that could not be associated by the demonstration of competitive inhibition with Mg2+, Na+, or K+ sites were classed as 4 non-specific inhibitors. These cations act at relatively low concentrations, and the concentrations of ions giving 50% inhibition of enzyme activity are correlated with the oxidation potential of the ion and with the binding constants to ethylenediamine, histidine, and imidazole. These results suggest that the non-specific ion inhibitors may react at one site—a histidine-like residue near the active center of the enzyme. 

The high affinity of the reuptake systems for monoamines, as measured by the Michaelis constant, is on the order of 0.1-0.4 pM the low affinity is about 10-fold higher. Specific uptake inhibitors for each monoamine have apparently been developed. Chlorimipramine and Lilly 110/40 are presumed to be specific for serotonergic uptake systems. Benztropine and amphetamine (an action separate from its ability to enhance catecholamine release) are thought to block specifically dopamine uptake and desmethyl-imipramine and cocaine are believed to inhibit norepinephrine uptake. 

Hence, the effect of a competitive inhibitor is to increase the apparent value of the Michaelis constant (Ka) by the factor (i+J/Jej). to reduce that of the specificity constant the same factor, and to leaveV, unchanged. 

In contrast to the competitive case, the effect of a noncompetitive inhibitor is to decrease the apparent value of the maximal velocity, Vi, by a factor proportional to (i+//iCii), to decrease the specificity constant, VJKb, by a factor proportional to (i+I/Ki ), and to change the Michaelis constant, Kb, by a factor proportional to (i+// Tig)/(i+//Aii). 

Comparison of the ordinary Michaelis-Menten relation with (5.108) shows that the inhibitor did not influence specific growth rate, vmgx, but the Michaelis-Menten constant was affected by the inhibitor and resulted in a constant, known as the apparent Michaelis constant. 

In this scheme, EOH is the enzyme, IX is the inhibitor (either a carbamate or an organophosphate). EOH(IX) is analogous to the Michaelis Menton comploc seen with the substrate reaction. EOI is the acyl-enzyme intermediate for carbamates or a phosphoro-enzyme intermediate for the organophosphates. The equilibrium constant for this reaction (K ) is defined as k /k and the phosphorylation or carbamylation constant is defined as k2- In this study 42)y ANTX-A(S) was found to be more specific for AChE than BUChE. The double reciprocal and Dixon plot of the inhibition of electric eel AChE indicated that the toxin is a non-competitive inhibitor decreases, k remains unchanged) (Figure 2). 

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. 

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

FIGURE 5.4 In this illustration of a competition experiment, a fixed concentration of radioligand, in the absence of inhibitor, produces specific binding of B0. The specific binding in the presence of a competitive inhibitor is denoted by Bt A constant amount of nonspecific binding is assumed to be present. The concentration of inhibitor that reduces specific binding by 50% is referred to as the IC50. 

---