Inhibition, of enzyme catalysis

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-... 

This article describes various approaches to inhibition of enzyme catalysis. Reversible inhibition includes competitive, uncompetitive, mixed inhibition, noncompetitive inhibition, transition state, and slow tight-binding inhibition. Irreversible inhibition approaches include affinity labeling and mechanism-based enzyme inhibition. The kinetics of the various inhibition approaches are summarized, and examples of each type of Inhibition are presented. 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

Metal ions (such as Zn + or Cu " ") at the trace level are often essential to biochemical reactions, for example, in catalysis, transport, or biosynthesis. However, at higher concentrations, accumulation of these ions in an organism can lead to unhealthy interactions such as biochemical redox processes and inhibition of enzyme activity. Therefore, the detection of metal cations is of great interest to many scientists [10,16,18,24]. 

Like other biocatalysts, RNA-cleaving ribozymes are highly specific for RNA and catalyze hydrolysis with high catalytic efficiency, undergo rapid turnover, and operate in a highly selective manner. In addition, inhibition of ribozyme catalysis has been observed for substrate analogs like enzymes and other biomacro-molecules, changes in ribozyme solvation and conformation have been observed when the inhibitor binds (Piccirilli et al. 1992). 

Since biological systems are dynamic, with many different processes taking place and many different substances present, buffers are necessary to prevent the kind of wide variation of pH that can inhibit proper enzyme catalysis. Thus, a proper pH aids in regulating the reaction rates associated with certain enzymes and maintaining them at levels appropriate for their particular functions. Two important biological buffers are the phosphate buffer system that regulates pH for the fluid inside cells and the carbonic acid buffer system that regulates pH for blood plasma. The chemical equations for these buffers are shown below for an aqueous solution. 

Negative catalysis may also occur, since several compounds are known that inhibit reactions. For example, some cations, notably Cu2+, catalyze the autoxidation of lipids chelating agents like citrate may greatly lower the activity of divalent cations, thereby decreasing the oxidation rate. Inhibition of enzymes is frequently observed. 

Preparation of the sample. Either semm or plasma (oxalated or heparinized) may be used for the determination. This may be freshly drawn blood from an animal. (Do not do this yourself your instructor will supply the sample.) See Chapter 1 for a discussion of the differences between semm, plasma, and whole blood. A 10- to 15-mL sample (20 to 30 mL whole blood) should be adequate for triplicate determinations by a class of 30 students. Fluoride should be added to prevent glycolysis, or breakdown of glucose, which can change the pH. The fluoride inhibits the enzyme catalysis causing glycolysis and stabilizes the pH for about 2 h. The tube used for collecting the sample can be rinsed with a solution of 100 mg sodium heparin plus 4 g sodium fluoride per 100 mL. The sample should be kept anaerobically, that is, stoppered to keep out atmospheric CO2. Since the analysis should be done on the day the blood is drawn, the solutions should be prepared ahead of time. 

Understand the basic kinetic equations of enzyme catalysis and inhibition... 

The properties of the carriers involved in both active and passive transport suggest that they are proteins. Apart from the fact that no other type of molecule has the necessary ability for specific recognition of the substance to be carried, carrier-mediated transport mechanisms show features that are reminiscent of enzyme catalysis, i.e. they are pH dependent, can be competitively inhibited by structurally similar compounds and poisoned by other compounds. Moreover, they show a relationship to the concentration of transported material that is essentially similar to that of substrate concentration on enzyme activity. It appears therefore that passive carrier-mediated transport may be a catalysed permeability in which the equilibrium of the reaction is not affected although the rate of attainment of equilibrium is greatly enhanced while in the case of active transport, there is a modification of the diffusion equilibrium as a result of the coupling of the transport process to an energy-yielding reaction. 

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

The activity of an enzyme can be modulated reversibly or irreversibly by inhibitors and inactivators. The kinetics of inhibition and/or inactivation provide valuable insights into the nature of essential and/or catalytic residues as well as the mechanism of enzyme catalysis. 

The Michaelis-Menten kinetic scheme, which involves a single substrate and a single product, is obviously the simplest type of enzyme catalysis. Equation (1.7) may hold for many mechanisms, but the mechanisms can be different from each other and the expression of kinetic parameters may also differ. When there is a substrate inhibition or activation due to the binding of a second substrate molecule, the Michaelis-Menten equation does not hold. 

Gertz 1. Likhtenshtein received his Ph.D. degree in 1963 at the N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences. The topic of his thesis was Oxidative Destruction and Inhibition of Polymers . Then his research interest moved to enzyme catalysis and he began his carrier at the Institute of Molecular Biology, Academy of Sciences. In 1965 Likhtenshtein returned to the Institute of Chemical Physics and was appointed on the position of the Head of Laboratory of Chemical Physics of Enzyme Catalysis. This Institute granted him the degree of Doctor... 

Substances that do not target the active site but display inhibition by allosteric mechanisms are associated with a lower risk of unwanted interference with related cellular enzymes. Allosteric inhibition of the viral polymerase is employed in the case of HIV-1 nonnucleosidic RT inhibitors (NNRTl, see chapter by Zimmermann et al., this volume) bind outside the RT active site and act by blocking a conformational change of the enzyme essential for catalysis. A potential disadvantage of targeting regions distant from the active site is that these may be subject to a lower selective pressure for sequence conservation than the active site itself, which can lower the threshold for escape of the virus by mutation. 

AOPP and AOA inhibit transaminase enzymes (39, 44) and other pyridoxylphosphate-dependent enzymes, presumably by interference with the carbonyl group of pyridoxyl phosphate (45). They apparently inhibit PAL by interaction with the carbonyl-like group involved in catalysis by PAL (36). AOA is not an effective PAL inhibitor for in vivo studies because of its lack of specificity that results in a relatively high degree of phytotoxicity (e.g. 

---