Enzyme covalent binding

Irreversible inhibition occurs with organophos-phorus insecticides and chemical warfare agents (see p. 437) which combine covalently with the active site of acetylcholinesterase recovery of cholinesterase activity depends on the formation of new enzyme. Covalent binding of aspirin to cyclo-oxygenase... 

Fig. 1. Immobilized enzymes. Covalent binding of an enzyme to an unsubstituted polysaccharide (e.g. Sepharaose) by the cyanogen bromide method.

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-... 

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

Wortmannin is a fungus-derived inhibitor of PI 3-kinase. The agent binds and inhibits the enzyme covalently and irreversibly. It is very potent and considered to be highly specific (IC5o in most cells in the low nanomolar range). 

Irreversible metabolic inhibition caused by covalent binding of the inhibitor to the enzyme after being metabolized by the same enzyme. The inhibitory effect remains after elimination of the inhibitor from the body. 

The first is cell injury (cytotoxicity), which can be severe enough to result in cell death. There are many mechanisms by which xenobiotics injure cells. The one considered here is covalent binding to cell macromol-ecules of reactive species of xenobiotics produced by metabolism. These macromolecular targets include DNA, RNA, and protein. If the macromolecule to which the reactive xenobiotic binds is essential for short-term cell survival, eg, a protein or enzyme involved in some critical cellular function such as oxidative phosphorylation or regulation of the permeability of the plasma membrane, then severe effects on cellular function could become evident quite rapidly. 

Not all enzyme inhibitors bind through reversible interactions. In some cases enzymes are inactivated by formation of covalent complexes with inhibitory molecules. 

Most poly(HA) depolymerases are inhibited by reducing agents, e.g., dithio-erythritol (DTT), which indicates the presence of essential disulfide bonds, and by serine hydrolase inhibitors such as diisopropyl-fluoryl phosphate (DFP) or acylsulfonyl derivates. The latter compounds covalently bind to the active site serine of serine hydrolases and irreversibly inhibit enzyme activity [48]. 

Fig. 6.14. Quenched activity based probe for the imaging of cathepsins. Upon covalent binding of the sensor to histidine and serine residues in the active site of the enzyme, the quencher is released and increased fluorescence indicates the now covalently labeled enzyme of interest. 

This process involves the suspension of the biocatalyst in a monomer solution which is polymerized, and the enzymes are entrapped within the polymer lattice during the crosslinking process. This method differs from the covalent binding that the enzyme itself does not bind to the gel matrix. Due to the size of the biomolecule it will not diffuse out of the polymer network but small substrate or product molecules can transfer across or within it to ensure the continuous transformation. For sensing purposes, the polymer matrix can be formed directly on the surface of the fiber, or polymerized onto a transparent support (for instance, glass) that is then coupled to the fiber. The most popular matrices include polyacrylamide (Figure 5), silicone rubber, poly(vinyl alcohol), starch and polyurethane. 

Some important factors that must be considered in the selection of the support for covalent binding are its capacity to bind the enzyme, as the linearity and the limit of detection of the sensing layers will be influenced by this value the mechanical and chemical stability of the support the efficiency of interaction with the analyte or the sample matrix the ease of preparation and the cost, regenerability and availability of the material. 

Covalent binding of chemical carcinogens to cellular macromolecules, DNA, RNA and protein, is wel1-accepted to be the first step in the tumor initiation process ( 1, 2). Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species which react with cellular macromolecules. Understanding the mechanisms of activation and the enzymes which catalyze them is critical to elucidating the tumor initiation process. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Colvin, M., Smolka, A., Rembaum, A., and Chang, M. (1988) The Covalent Binding of Enzymes and Immunoglobulins to Hydrophilic Microspheres in Microspheres-. Medical and Biological Applications, pp. 1-13. CRC, Boca Raton, FL. 

Varanasi, U. and D.J. Gmur. 1980. Metabolic activation and covalent binding of benzo[a]pyrene to deoxyribonucleic acid catalyzed by liver enzymes of marine fish. Biochem. Pharmacol. 29 753-762. 

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