Specificity, enzymes

Enzymes can be either totally specific for a given substrate, to such an extent that they will not tolerate any structural or configurational changes in the substrate, or they can be broadly specific for a given type of functional group, and they will still operate on substrates with structural variations around that functional group. An example of the former is the enzyme aspartase, Asp, which catalyzes both the addition of ammonia to fumaric acid and the elimination of ammonia from L-aspartic acid by the following reactions  

Asp will not catalyze the addition of NH3 to the cis acid, maleic acid, and it will not catalyze the elimination of NH3 from D-aspartic acid. Chymotrypsin, on the other hand, is an example of an enzyme with broad activity, and it can catalyze 

Enzyme specificity refers to the strict limitation of the action of each enzyme to one substance or to a small number of closely related substances. Thus enzyme specificity bestows enzymes with abilities to discriminate among great number of metabolites in the cell, which is the essence of ordered metabolisms of living matter. There are several types of enzyme specificity  

Reaction specificity Many enzymes promote the same reaction acting on a small number of closely related substances, usually with a particular functional group such as phosphase, which mediates hydrolysis of phosphomonoesters and hexose kinase, which phosphorylates aldohexoses  

Stereospecificity Enzymes are also steiicaUy specific when acting on substrates that are stereoisomeric, i.e. isomers in which the atoms are oriented differently in space. The presence of a chiral (asymmetric) center (carbon) in a molecule gives rise to an enatiomeric pair, D and L or R and S (Cahn et al, 1966). Stereospecific enzymes that act on only one enantiomer but not the other are known as chiral stereospecificity. For example, o-lactate dehydrogenase oxidizes only D-lactate to pyruvate and L-lactate dehydrogenase, its c-enantiomer  

Stereospecificity may also apply to enzymes acting on the substrates containing double bonds, which give rise to geometric isomers, cis and trans or E and Z. For example, fumerase specifically catalyzes trans hydration of fumerate (trans-butenedioic acid)  

One of the most subtle stereospeciflcity of enzymes relates to their ability to distinguish between two identical atoms/groups (proR versus proS) bonded to a carbon atom in prochiral stereospeciflcity (Hanson and Rose, 1975). For example, yeast fermentative glycerol kinase catalyzes phosphorylation of only one (proS) of the two chemically identical primary hydroxyl groups to yield L-glycerol-3-phosphate  

Fischer was intrigued by the fact that emulsin caused hydrolysis of both / -glucosides and / -galactosides but had no effect on either the a- or / -xylo-sides (33). Since, at the time, Fischer expected glycosides to be furanosides, he suggested that both the enzymes required the presence of a free hydroxyl group at position 5 of a hexoside. 

At this point Fischer concluded that the enzymes, in terms of the configurations of the substrates, are as fastidious as yeast and other organisms. He then returned to the above-mentioned hypothesis that he and Thierfelder had proposed (30) and concluded (32) that the protein substances known as invertin and emulsin, like the substrates whose hydrolyses they effected, were asymmetrically formed molecules. On the basis of this consideration, he came to the momentous lock and key concept for enzyme activity and commented as follows  

The restricted effects of the enzymes may therefore be explained by the assumption that the approach of the molecules that cause the chemical process can occur only in the case ofa similar geometric shape. 

To use a picture, / would like to say that enzyme andglucoside have to fit to each other like a lock and key in order to exert a chemical effect on each other. 

Emil Fischer developed a strong interest in the structural requirements for enzyme activity as the result of effects of changes in the structures of the a-and -methyl glucosides on their properties as substrates for the enzymes invertin and emulsin, which, as we have seen, he had shown to be a- and yS-glucosidases, respectively. As already mentioned, he was fascinated in 1895 by the fact that emulsin had no effect on either the a- or /2-methyl xylosides (33). In a 1912 publication with Karl Zach (34), he reported that -methyl 6-deoxyglucoside was hydrolyzed by emulsin and wrote  

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The search for new antivkal agents is ongoing and extensive not all vimses have been included here, and new vimses pathogenic to humans will continue to be identified. Novel nucleosides as weU as nonnucleosidic compounds which possess greater potency and enzymic specificity for the future treatment of both acute and persistent human vkal infection will continue to be discovered. 

Enzymes are used as feed digestibiUty enhancers for chicken and pigs. They must comply with purity specifications comparable to food-grade enzyme specifications. European Community (EC) guidelines for the assessment of additives in animal nutrition are being revised to make them appHcable for enzymes. Upon completion of these guidelines, the regulatory status of feed enzymes will be estabUshed in EC directive 70/524/EEC. 

Carter, P., Wells, J.A. Engineering enzyme specificity by "substrate-assisted catalysis." Science 237 394-399, 1987. 

Pancreatic RNa.se is an enzyme specific for cleavage where a pyrimidine ba.se lies to die 3 -side of die pho.sphodie.ster it acts endo. The products are oligonucleotides widi pyrimidine-3 -P04 ends ... 

Pioneering enzyme specificity studies at the turn of the century by the great organic chemist Emil Eischer led to the notion of an enzyme resembling a lock and its particular substrate the key. This analogy captures the essence of the specificity that exists between an enzyme and its substrate, but enzymes are not rigid templates like locks. 

The protein from D. desulfuricans has been characterized by Mbss-bauer and EPR spectroscopy 224). The enzyme has a molecular mass of approximately 150 kDa (three different subunits 88, 29, and 16 kDa) and contains three different types of redox-active centers four c-type hemes, nonheme iron arranged as two [4Fe-4S] centers, and a molybdopterin site (Mo-bound to two MGD). Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. 

Mutter M, Beldman G, Schols HA, Voragen AGJ (1994) Rharrmogalacturonan a-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rharruiosyl unit in rhamnogalacturonan regions of pectin. Plant Physiol 106 241-250... 

The EMSIL method may prove useful in cytochemical applications where lack of substrate immunogenicity precludes antibody generation, or for which enzymes specific to the... 

Several scouting experiments were performed to find the best pH conditions. Figure 3 reports the ratio between the PG specific activity measured after the purification procedure (ASf) and the initial PG specific activity (ASi). At pH 3.5, the microspheres are able to remove from the broth the major part of the protein without PG activity, thus providing a four time increase of the enzyme specific activity. The purified PG from Kluyveromyces marxianus was immobilised following the above procedure. Batch reactions in the packed bed reactor were done to evaluate the biocatalyst stability. After an initial loss, due to enzyme release, the residual PG activity reaches a plateau value corresponding to about 40% of the initial activity. Probably, some broth component interfered during the immobilisation reaction weakening the protein-carrier interactions. 

Parales JV, RE Parales, SM Resnick, DT Gibson (1998) Enzyme specificity of 2-nitrotoluene 2,3-dioxygenase from Pseudomonas sp. strain JS42 is determined by the C-terminal region of the a subunit of the oxygenase component. / Sacfeno/ 180 1194-1199. 

Enzymes specifically cleave the backbone structure of the thickeners and eventually of the fluid loss additive. They offer several advantages to other... 

Organic solvent can affect the enzyme specificity [76]. Authors have indicated that transesterification of l,4-butyloxy-2-octylbenzene and butanol in presence of lipases from Pseudomonas can produce two different products when using hydrophilic (acetonitrile) or hydrophobic (toluene) solvents. Zaks and Klibanov [16], demonstrated that subtilisine and a-chymotrypsine specificites can be changed as a function of solvent types. This is true for a limited number of biocatalysts. 

The review articles by Schramm (1998, 2003) provide a number of examples of the successful application of this protocol to the design of enzyme-specific transition state-like inhibitors. Among these, the transition state inhibitors of human purine nucleoside phosphorylase (PNP) are particularly interesting from a medicinal chemistry perspective, as examples of these compounds have entered human clinical trials for the treatment of T-cell cancers and autoimmune disorders. 

The lack of target enzyme specificity is a critical liability for the use of affinity labels as drugs. The inherent chemical reactivity of these compounds almost ensures that... 

Enzyme specificity is often explained in terms of the geometric configuration of the active site of the enzyme. The active site includes the side chains and peptide bonds that either come into direct contact with the substrate or perform some direct function during catalysis. Each site is polyfunctional in that certain parts of it may hold the substrate in a position where the other parts cause changes in the chemical bonding of... 

The specificity of several of the enzymes identified in the 4S pathway of different organisms has been studied. In case of the DBT desulfurizing enzymes, little difference is expected in the specificity of the enzymes, say DszA, from different Rhodococcus strains found to date. This is essentially because the DNA sequence for the enzymes investigated so far has been the same. The difference in the specificity observed with whole cell assays is essentially due to the differences in substrate intake via the cell membrane and not necessarily due to a difference in the intrinsic enzyme specificity. It has been found that while isolated enzyme DszC (from KA2-5-1) can desulfurize up to 4,6 dipropyl DBT, whole cells cannot, indicating substrate transport as limiting factor. 

While the use of FRET probes in vitro is inflicted with a limited number of complications, the use in biological samples or in living cells needs much more careful considerations since factors such as enzyme specificity, cell toxicity, and spatio-temporal resolution usually play an important role. 

SCHEME 2 Schematic illustration of analytical mechanism of (a) first-, (b) second-, and (c) third-generation 02 biosensors. Note that the reactions shown in (b) and (c) are bi-directional since SODs are enzymes specifically catalyzing the 02 dismutation, i.e. oxidation into 02 and reduction into H202. 

Gottschalk, Alfred, Principles Underlying Enzyme Specificity in the.Domain of Carbohydrates, V, 49-78... 

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