Irreversible enzyme inactivators

Figure 8.1 Typical enzyme reaction progress curve in the presence of an irreversible enzyme inactivator, highlighting the initial velocity region (v ) and the fact that the terminal velocity (vs) is zero for such compounds.

Figure 8.2 Mechanisms of irreversible enzyme inactivation. (A) Nonspecific affinity labeling, (B) quiescent affinity labeling, and (C) mechanism-based inactivation.

Hence, for any irreversible enzyme inactivator, we can quantify the effectiveness of inactivation using the second-order rate constant kanJKi. This constant thus becomes the key metric that the medicinal chemist can use in exploring the SAR of enzyme inactivation by a series of compounds. In terms of individual rate constants, the definitions of both nact and A) depend on the details of the mechanisms of inactivation, as will be described below. 

An important point to realize here is that attempts to quantify the relative potency of irreversible enzyme inactivators by more traditional parameters, such as IC50 values, are entirely inappropriate because these values will vary with time, in different ways for different compounds. Hence the SAR derived from IC50 values, determined at a fixed time point in the reaction progress curve, is meaningless and can be misleading in terms of compound optimization. Unfortunately, the literature is rife with examples of this type of inappropriate quantitation of irreversible inactivator potency, making meaningful comparisons with literature data difficult, at best. 

However, an important problem arises during the peroxidative removal of phenols from aqueous solutions PX is inactivated by free radicals, as well as by oligomeric and polymeric products formed in the reaction, which attach themselves to the enzyme (Nazari and others 2007). This suicide peroxide inactivation has been shown to reduce the sensitivity and efficiency of PX. Several techniques have been introduced to reduce the extent of suicide inactivation and to improve the lifetime of the active enzyme, such as immobilization. Moreover, Nazari and others (2007) reported a mechanism to prevent and control the suicide peroxide inactivation of horseradish PX by means of the activation and stabilization effects of Ni2+ ion, which was found to be useful in processes such as phenol removal and peroxidative conversion of reducing substrates, in which a high concentration of hydrogen peroxide may lead to irreversible enzyme inactivation. 

From the above scheme, it is clearly not necessary that every conversion of El to E results in irreversible enzyme inactivation. The partition ratio for an inhibitor is the ratio of product released to enzyme inactivated. Mathematically, the partition ratio is equal to h/ki, with a ratio of zero indicating that every turnover of inhibitor molecule results in enzyme inactivation. The partition ratio does not depend on the concentration of the inactivator, hut rather on the reactivity of I, its proximity to a suitable target on the enzyme with which it might react, and its rate of diffusion away from the active site. 

The mechanisms of irreversible enzyme inactivation at 100C, Science 1985, 228, 1280-1284. 

A new and elegant approach to specific irreversible enzyme inactivation is the use of inhibitors possessing latent reactive functionalities which are unmasked at the enzyme s active site as a result of the normal catalytic turnover. Such an inhibitory process is described by the following equation ... 

Treatment of this enzyme with substrate and sodium boro-hydride leads to irreversible enzyme inactivation via in situ reduction of the enzyme-bound imine intermediate by boro-hydride, which indicates that a covalent link is formed. As mentioned, the pKa of this lysine group is abnormally low at 5.9, which is sufficiently low for it to act as a nucleophile at pH 7 (8). 

Suppose that you want to keep a solution of an enzyme in an aqueous buffer at —30°C without freezing (because the freeze concentration would lead to irreversible enzyme inactivation). How can this be achieved Assume the number of catalytic impurities at —30°C to be 1011 m-3. 

As previously described, irreversible enzyme inhibition is defined as time-dependent inactivation of the enzyme, which implies that the enzyme has, in some way or form, been permanently modified, because it can no longer carry out its function. This modification is the result of a covalent bond being formed with the inhibitor and some amino acid residue in the protein. Furthermore, this bond is extremely stable and, for all practical purposes, is not hydrolyzed fo give back the enzyme in its original state or structure. In most examples of irreversible inhibition, a new enzyme must be generated through gene transcription and translation for the enzyme to continue its normal catalytic action. Basically, there are two types of irreversible enzyme inhibitors, the affinity labels or active site-directed irreversible inhibitors and the mechanism-based irreversible enzyme inactivators. 

Mechanism-Based Irreversible Enzyme Inactivators Overview... 

Pectin methylesterase (EC 3.1.1.11) causes the flocculation of pectic acid (cf. 2.7.2.2.13) in orange juices and reduces the consistency of tomato products. In orange juice, irreversible enzyme inactivation reaches 90% at a pressure of 600 MPa. Even though the enzyme in tomatoes is more stable, increasing the temperature to 59-60 °C causes inactivation at 400 MPa and at 100 MPa after the removal of Ca + ions. 

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