Enzymes decreasing Active sites

Elucidating Mechanisms for the Inhibition of Enzyme Catalysis An inhibitor interacts with an enzyme in a manner that decreases the enzyme s catalytic efficiency. Examples of inhibitors include some drugs and poisons. Irreversible inhibitors covalently bind to the enzyme s active site, producing a permanent loss in catalytic efficiency even when the inhibitor s concentration is decreased. Reversible inhibitors form noncovalent complexes with the enzyme, thereby causing a temporary de-... 

Competitive inhibition occurs when chemicals compete for the space at another enzyme s active site and this results in decreased apparent affinity (i.e., increased Km), and therefore reduces the rate of metabolism at lower substrate concentrations. Whether the competitive inhibitor is another substrate or strictly an inhibitor, the mathematical description is the same (Table 2.2). The inhibition constant (K) of a competing substrate should have the same value as its Km for the enzyme involved (Segel 1975) since both parameters reflect the chemical s affinity to the enzyme site of action. 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

To this end, the spin labeling technique was used to probe the active site structure of LADH in various solvent systems. The spin label, SL-1, alkylated cysteine 46 (12), an amino acid in the active site of LADH that normally serves as a ligand to the catalytic zinc ion. LADH is a dimer, and each monomeric subunit contains one firmly-bound catalytic zinc ion. The position of enzyme-bound SL-1 was estimated by the spin label-spin probe technique (14.15). Cobalt (II) was employed as the spin probe, and the catalytic zinc in the enzyme s active site was replaced by Co + according to the procedure of Sytkowski and Vallee (16). The EPR spectrum of enzyme-bound SL-1 was then measured before and after replacement of the active-site zinc by cobalt from the decrease in spectral amplitude the average nitroxide-metal distance was determined to be 4.8 1.5 A (17). 

Reversible inhibition may be further subclassified as to its competitive or noncompetitive characteristics. Competitive inhibition occurs when the inhibitor competes with the natural substrate at the enzyme s active site. The reversible enzyme-inhibitor complex formed thus prevents, or decreases, access to the active site by the substrate. Equation 2.3 summarizes these events, where S is the substrate concentration and P is the product, and ultimately an effect. The degree to which the rate of P formation is affected depends on the concentration of inhibitor (7) and the dissociation rate of El represented by Kt. Smaller numerical values for Kt indicate stronger inhibitor-enzyme binding. In the competitive state inhibition can be overcome by increased levels of a substrate. 

Superimposed on the allosteric regulation is inhibition by adenylylation of (addition of AMP to) Tyr , located near the enzyme s active site (Fig. 22-7). This covalent modification increases sensitivity to the allosteric inhibitors, and activity decreases as more subunits are adenylylated. Both adenylylation and deadenylylation are promoted by adenylyltransferase (AT in Fig. 

There are many compounds that affect enzymatic reaction rates due to different mechanisms. Activators, such as cofactors and coenzymes, are compounds that bind with the enzyme and increase reaction rates. On the other hand, inhibitors are compounds that bind to the active site and reduce the rate by negatively influencing the catalytic properties of the enzyme s active sites (Panesar et al., 2010). In addition, an inhibitor can also bind at sites other than the enzyme s active site. For example, on the reverse, resnlting in conformational changes in the active site and a decrease in catalytic activity. 

We saw in Section 3.15 that statins (Lipitor, Zocor, Mevacor) lower serum cholesterol levels. These drugs are competitive inhibitors for the enzyme that reduces hydroxymethylglutaryl-CoA to mevalonic acid (page 1197). Recall that a competitive inhibitor competes with the substrate for binding at the enzyme s active site (Section 24.7). Decreasing the concentration of mevalonic acid decreases the concentration of isopentenyl pyrophosphate, so the synthesis of aU terpenes, including cholesterol, is decreased. As a consequence of diminished cholesterol synthesis, the liver forms more LDL receptors— the receptors that help clear LDL from the bloodstream. Recall that LDL (low-density lipoprotein) is the so-caUed bad cholesto-ol (Section 3.15). 

Inhibition The decrease of the rate of an enzyme-catalyzed reaction by a chemical compound including substrate analogues. Such inhibition may be competitive with the substrate (binding at die active site of die enzyme) or non-competitive (binding at an allosteric site). 

Bell-shaped activity versus pH profiles arise from two separate active-site ionizations, (a) Enzyme activity increases upon deprotonation of (b) Enzyme activity decreases upon deprotonation of A-H. (c) Enzyme activity is maximal in the pH range where one ionizable group is deprotonated (as B ) and the odier group is protonated (as A-H). 

Ornithine decarboxylase is a pyridoxal dependent enzyme. In its catalytic cycle, it normally converts ornithine (7) to putrisine by decarboxylation. If it starts the process with eflornithine instead, the key imine anion (11) produced by decarboxylation can either alkylate the enzyme directly by displacement of either fluorine atom or it can eject a fluorine atom to produce viny-logue 12 which can alkylate the enzyme by conjugate addidon. In either case, 13 results in which the active site of the enzyme is alkylated and unable to continue processing substrate. The net result is a downturn in the synthesis of cellular polyamine production and a decrease in growth rate. Eflornithine is described as being useful in the treatment of benign prostatic hyperplasia, as an antiprotozoal or an antineoplastic substance [3,4]. 

One of the most efficient plasmin inhibitor is a2-PI (70 kDa), which is synthesized by the liver, secreted into the blood circulation, where its concentration is 1 pM. It rapidly forms equimolar complex with plasmin, and in this complex, the active site of the enzyme is irreversibly blocked. The complex, thereafter, is removed by the liver. It is remarkable that when plasmin is bound to its substrate (fibrin), it is protected against its primarily inhibitor, a2-PI the rate of inactivation decreases by 400-fold (Fig. 4) [3]. 

Because mechanism-based inactivators behave as alternative substrates for the enzyme, they must bind in the enzyme active site. Binding of a mechanism-based inactivator is therefore mutually exclusive with binding of the cognate substrate of the normal enzymatic reaction (we say cognate substrate here because for bisubstrate reactions, the mechanism-based inactivator could be competitive with one substrate and noncompetitive or uncompetitive with the other substrate of the reaction, depending on the details of the reaction mechanism). Thus, as the substrate concentration is increased, the observed rate of inactivation should decrease (Figure 8.10) as... 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Inhibition Effects in Enzyme Catalyzed Reactions. Enzyme catalyzed reactions are often retarded or inhibited by the presence of species that do not participate in the reaction in question as well as by the products of the reaction. In some cases the reactants themselves can act as inhibitors. Inhibition usually results from the formation of various enzyme-inhibitor complexes, a situation that decreases the amount of enzyme available for the normal reaction sequence. The study of inhibition is important in the investigation of enzyme action. By determining what compounds behave as inhibitors and what type of kinetic patterns are followed, it may be possible to draw important conclusions about the mechanism of an enzyme s action or the nature of its active site. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

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