Enzymes active sites, nature

Gilson, M.K. and Honig, B.H. (1987) Calculation of the electrostatic potentials in an enzyme active site. Nature, 330, 84-86. 

Gilson, M. and B. Honig. (1987). Calculation of Electrostatic Potentials in an Enzyme Active Site. Nature. 330 84. 

The outstanding inclusion ability and the carboxylic functions of host I raised the idea of co-erystallizing it with imidazole (Im) which, due to its versatile nature 114), is one of the frequently used components in enzyme active sites, generally presented by histidine. Formally, a system made of imidazole and an acid component may mimic two essential components of the so-called catalytic triad of the serine protease family of enzymes the acid function of Aspl02 and the imidazole nucleus of His57 115) (trypsin sequence numbering). The third (albeit essential) component of the triad corresponding to the alcohol function of Seri 95 was not considered in this attempt. This family of enzymes is of prime importance in metabolitic processes. 

Diacylglycerol has long been known to be a weak competitive inhibitor of PLC/fc, whereas phosphorylcholine shows very little inhibition [40, 49, 116]. Recent kinetic assays of PLCB(. activity in the presence of DAG indicate that it is a competitive inhibitor with a Kl of the order of 10 mM, whereas phosphorylcholine was found to be an extremely weak (K = 30-50 mM), mixed inhibitor of PLC/J( [34]. Because diacylglycerol is a competitive inhibitor of the enzyme, the nature of the catalytic cycle dictates that it must be the last product to leave the enzyme active site. 

When binding of a substrate molecule at an enzyme active site promotes substrate binding at other sites, this is called positive homotropic behavior (one of the allosteric interactions). When this co-operative phenomenon is caused by a compound other than the substrate, the behavior is designated as a positive heterotropic response. Equation (6) explains some of the profile of rate constant vs. detergent concentration. Thus, Piszkiewicz claims that micelle-catalyzed reactions can be conceived as models of allosteric enzymes. A major factor which causes the different kinetic behavior [i.e. (4) vs. (5)] will be the hydrophobic nature of substrate. If a substrate molecule does not perturb the micellar structure extensively, the classical formulation of (4) is derived. On the other hand, the allosteric kinetics of (5) will be found if a hydrophobic substrate molecule can induce micellization. 

A computer graphics facility was then used with available crystal data, molecular orbital and molecular mechanics calculations/ infra-red and n.m.r. studies to construct a three dimensional model of the target enzyme active site (a cytochrome P-450) designed specifically to accommodate both the natural substrate (24 methylene 24 25 dihydrolanosterol) and these known antagonists in their minimum or low energy forms. 

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

The ability of cyclic ethers to complex biologically important alkylammonium cations makes the choice of crown ethers as enzyme binding site models a natural one. In recent years a number of molecules containing both a crown ether-based substrate binding site and a potentially reactive group have been prepared as models for enzyme active sites (79PAC979, B-82MI52100). 

In general, hydrolytic enzymes can be classified based on the type of reaction catalyzed, the nature of the enzyme active site, and/or evolutionary relationships among enzymes, as derived from primary sequence data. Among proteases, gross functional distinctions are made between serine proteases, aspartic proteases, cysteine proteases, and metalloproteases. Each of these groups includes a diverse range of enzymes of distinctive size and structure for example, an aminopeptidase isolated from B. lichenformis was found to have a molecular mass of 34,000, whereas an E. coli aminopeptidase had a mass of 400,000 (Rao et al., 1998). 

Several general approaches have been used to measure the activities of extracellular enzymes in aquatic systems. These methods typically measure a potential activity, inasmuch as a substrate added to a sample to measure enzyme activity is in competition with naturally occurring substrates (whose concentration is usually unknown) for enzyme active sites. The most commonly applied method involves a small substrate proxy, typically consisting of a monosaccharide or an amino acid covalently linked to a small fluorophore substrates frequently used include methyumbellifery- (MUF-) monosaccharides and 4-methyl-coumainylamide (MCA)- amino acids. Upon hydrolysis of the bond between the monomer and the fluorophore, the fluorophore becomes fluorescent, and hydrolysis is measured as an increase in fluorescence signal with time (Hoppe, 1983 Somville and Billen, 1983). 

FIGURE 3 Several hypothetical scenarios which might explain resistance of organic macromolecules to extracellular enzymatic hydrolysis (a) natural substrates are not a good fit for enzyme active sites, perhaps because of biological or chemical modifications (b) specific substrates are too dilute to induce enzymes under most circumstances (c) substrates are physically protected from hydrolysis (e.g., Mayer, 1994 Keil et al., 1994). Enzymes may also be complexed, hindering their activities (Wetzel, 1993). 

Enzyme inhibitors Inhibitors are substances that lower the activity of enzymes, reducing the speed of their reactions. Some inhibitors can even completely stop enzyme-catalyzed reactions. They are found naturally, but are also produced artificially as drugs, pesticides, and other substances. The most successful inhibitors are those that have a structure very similar to that of a substrate, so that they can bind to enzyme active sites. If the active sites are occupied by an inhibitor, the enzyme is no longer available to bind its real substrate. Other inhibitors do not look like substrates. Instead, they bind on an enzyme so as to distort its shape the geometry of the active site is changed so that the substrate no longer fits. 

Sokalski WA, Kedzierski P, Grembecka J (2001) Ab initio study of the physical nature of interactions between enzyme active site fragments in vacuo. Phys Chem Chem Phys 3 657-663... 

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