Specificity substrate

The characteristics of immobihzed enzymes, such as substrate specificity, optimum pH of enzyme reaction. Km, the maximum reaction rate and optimum temperature, etc, will differ from those of the free enzyme, because of the different immobihzation processes. 

The activities of allosteric enzymes (cf. 2.5.1.3) are affected by specific regulators or effectors. Thus, the activities of such enzymes show an additional regulatory specificity. 

The substrate specificity of enzymes shows the following differences. The occurrence of a distinct functional group in the substrate is the only prerequisite for a few enzymes, such as some hydrolases. This is exenqtlified by nonspecific lipases (cf. Table 3.21) or peptidases (cf. 1.4.5.2.1) which generally act on an ester or peptide covalent bond. 

More restricted specificity is found in other enzymes, the activities of which require that the substrate molecule contains a distinct structural feature in addition to the reactive functional group. Examples are the proteinases tr3fpsin and chymotrypsin which cleave only ester or peptide bonds with the carbonyl group derived from lysyl or arginyl (trypsin) or t3rosyl, phenylalanyl or tryptophanyl residues (ch3miotr3fpsin). Many enzymes activate only one single substrate or preferentially catalyze the conversion of one substrate while other substrates are converted into products with a lower reaction rate (cf. ex- 

Catalysis by cyclodextrins often shows specificity which is characteristic of enzymatic catalysis. Here, the specificities are divided into 3 categories (1) substrate specificity, in which subtle changes in the structure of the substrates have large effects on the catalysis (2) product specificity, in which the products of the catalyzed reactions are highly selective and (3) d,l specificity, in which enantiomeric recognition is made by a-cyclodextiin. 

The most striking specificity by cyclodextrins with respect to the substrate is found in the hydrolyses of phenyl acetates. As shown in Table 4, the magnitudes of the acceleration by a-cyclodextrin for weto-substituted compounds are 29-, 

and 13-fold larger than those for para-substituted compounds, for methyl, /eri-butyl, nitro, and carboxyl substitution, respectively. Similar results are obtained also for /8-cyclodextrin. 

The larger magnitude of the acceleration of the cleavage of mera-substituted 

Catalytic rate constants and acceleration in the a-cyclodextrin-catalyzed hydrolyses of phenyl acetates  

Except for studies involved with characterization of the enzyme, there is little relevant material in the literature concerning the mechanism of the reaction. The incorporation of from water into isopentenyl pyrophosphate and dimethylallyl pyrophosphate has been determined. The exchange was substantially faster with isopentenyl pyrophosphate and the label was located primarily on C-4. The results were interpreted in terms of a mechanism in which a carbonium ion, formed by protonation, partitions between the two substrates and a covalent adduct with the enzyme (Fig. 9). A covalent substrate adduct was reported, but this undoubtedly resulted as an artifact of the experimental procedures used [6]. 

Enzymes are particularly valuable for the production of enantiomerically pure compounds, as shown in examples throughout this book. However, the narrow range of substrates that some enzymes accept and the less than impressive enantioselectivities exhibited by others often frustrate attempts to develop new synthetic applications and to commercialize existing ones. Directed evolution can efficiently tune substrate specificity and catalytic efficiency towards non-natural substrates it can also tailor enantioselectivity, as illustrated in the examples below. 

Zhang et al. evolved a fucosidase from a galactosidase1 61. Seven rounds of DNA shuffling and screening using a chromogenic fucose substrate yielded a mutant with 66-fold increase in fucosidase activity. Kinetic analysis of the purified enzyme revealed a 10- to 20-fold increase in kcat/Km for the fucose substrate and a 50-fold decrease for galactose (a total of 1000-fold increased substrate specificity for fucose). 

Kumamaru et al. recombined two biphenyl dioxygenases (96% identical) and visually screened for mutants whose substrate range differed from the parents . These mutants degraded various biphenyl compounds more efficiently and also exhibited oxygenation activity for single-ring aromatic compounds on which neither parent was active 

Aspartate aminotransferase catalyzes amino group transfer between acidic amino 

Fructose 1,6 bisphosphate (FBP) is an allosteric activator of the thermostable l-2-hydroxyacid dehydrogenase from B. stearothermophilus, which might be useful for the asymmetric synthesis of chiral compounds. Since FBP is quite expensive, Allen and Holbrook wished to create an FBP-independent variant1208. Three rounds of shuffling and screening produced a mutant L-2-hydroxyacid dehydrogenase with three amino acid substitutions that is almost as active in the absence of FBP as the wild-type is in its presence. 

PS-PLAi reacts specifically with PS and 1-acyl-lysoPS [20, 24]. It can not appreciably hydrolyze any other phospholipids including phosphatidyl-D-serine. The enzyme is not a type of phosphobpase B because it exclusively hydrolyzes an acyl residue bound at sn-1 position of either PS or 1-acyl-lysoPS. Incubation of the en- 

The kinetic parameters of the deiodination of different iodothyronines and their sulfate conjugates by rat liver microsomes as determined in this laboratory are summarized in Table II. Although these reactions have been carried out in the presence of DTT, the Vmm/Km ratio is thought to be a cofactor-independent indicator of the efficiency of the catalytic process [8] (see also Section 2.4). 

Reverse T3 is the preferred sustrate for the type I deiodinase the efficacy of its ORD to 3,3 -T2 is at least 500-fold higher than the deiodination of T4 and T3 [32], Reverse T3 is converted quantitatively to 3,3 -T2, and no evidence exists for the hepatic production of 3 5 -T, [32,33], T4 undergoes either ORD to T3 or IRD to rT3 but little of the latter is recovered as it is rapidly further degraded to 3,3 -T2 [32], The kinetics of T4 IRD have, therefore, been estimated by summation of the rT3 and 3,3 -T2 productions [32]. Production of 3,3 -T2 by IRD of T3 is a relatively slow process while ORD of T3 to 3,5-T2 has not been observed in liver. 

A recently recognized property of the type I deiodinase is its particular activity towards sulfated iodothyronine substrates [19,20,34], This was first discovered in 

Substrate specificity of rat liver type I iodothyronine deiodinase 

Kinetic parameters were determined using liver microsomes from euthyroid rats in 0.1 M phosphate (pH 7.2), 2 mM EDTA and 3-5 mM DTT. Km is expressed in /uM and Vmiis in pmol/min per mg protein. ND, not detectable. Data are taken from Refs. 20, 32 and 34. 

All known fungal nitrilases exhibit high relative activities towards benzonitrile and its m- and p-substituted derivatives. Therefore, according to the nitrilase classification [46] they belong to aromatic nitrilases. However, almost all fungal nitrilases also hydrolyze aUphatic nitriles, albeit at lower relative rates. 

The substrate specificities of all four biochemically characterized fungal nitrilases and two enzymes from rhodococci (an aromatic and an aUphatic nitrilase) are compared in Table 14.3. Only a few nitriles were examined as substrates of aU these enzymes. Moreover, the comparison of substrate specificity is difficult in some cases as different activity assays have been used for different enzymes. For instance, the determination of activity by ammonia measurement did not reflect potential amide formation. Nevertheless, common patterns can be found in substrate preferences of aromatic nitrilases. 

Differences between the reactivities of meta- and para-isomers also seem to reflect the electron densities on the cyano group [14]. For instance, a lower electron density may be expected on the cyano group of 1,4-dicyanobenzene in comparison with 1,3-dicyanobenzene and the much greater reactivity of the former compound is in accordance with this expectation. 

Nitrilases are largely very sensitive to steric hindrances by the substituent or heteroatom at the ortho-position, 2-substituted benzonitriles or 2-cyanopyridine being rarely hydrolyzed at acceptable rates. 

4-Cyanopyridine is a better substrate than 3-cyanopyridine for aU the examined nitrilases. The difference between the reactivities of these compounds can also be rationalized by a lower electron density on the cyano group of 4-cyanopyridine. 

1988) and specific chemical inhibitors such as KN-62 and KN-93. KN-62 inhibits CaM-kinase II with a K of 

9 (xM with no significant inhibitory effects on MLCK, cAMP-dependent protein kinase, or PKC (Tokumitsu et al., 1990). The mechanism of inhibition by KN-62, and a more water-soluble derivative KN-93 (Sumi et al., 1991), appears to be by competitive inhibition of calmodulin binding. Consequently, the autophosphorylated form of the kinase is not affected by the inhibitor (Tokumitsu et al., 1990). KN-62 has been used in intact PC-12 cells to inhibit CaM-kinase II auto-phosphorylation and generation of autonomous activity following stimulation by ionophores or KCl depolarization (Tokumitsu et al., 1990). 

FIGURE 5 (A) Autophosphorylation of CaM-kinase II 8-subunits in cultured rat aortic VSM cells. CaM- 

Dissociation of the endogenous Ca +ZCaM-dependent kinase activity and caldesmon was ultimately achieved by treatment of a myofibrillar fraction (from avian gizzard) with 30 mM Mg + and resolution by anion-exchange chromatography (Ikebe et al., 

The purified kinase was concluded to be a smooth muscle isozyme of CaM-kinase II based on its catalytic properties (using caldesmon as the primary 

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Microorganisms and their enzymes have been used to functionalize nonactivated carbon atoms, to introduce centers of chirahty into optically inactive substrates, and to carry out optical resolutions of racemic mixtures (1,2,37—42). Their utifity results from the abiUty of the microbes to elaborate both constitutive and inducible enzymes that possess broad substrate specificities and also remarkable regio- and stereospecificities. 

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

The TK-catalyzed reaction requires the presence of thiamine pyrophosphate and Mg " as cofactors. Although the substrate specificity of the enzyme has not been thoroughly investigated, it has been shown that the enzyme accepts a wide variety of 2-hydroxyaldehydes including D-glyceraldehyde 3-phosphate [591-57-1], D-glyceraldehyde [453-17-8], D-ribose 5-phosphate /47(9(9-2%/7, D-erythrose 4-phosphate and D-erythrose [583-50-6] (139,149—151). 

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiendy reduces all simple four- to nine-membered cycHc ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aUphatic acycHc ketones (C-4—C-10) (103,104) can be efficiendy achieved by alcohol dehydrogenase isolated from Thermoanaerohium hrockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse Hver and T. hrockii... 

Perhaps the biggest impact on the practical utilization of enzymes has been the development of nonaqueous enzymology (11,16,33,35). The use of enzymes in nonaqueous media gready expands the scope of suitable transformations, simplifies thek use, and enhances stabiUty. It also provides an easy means of regulation of the substrate specificity and regio- and enantioselectivity of enzymes by changing the reaction medium. 

MAO is known to occur in at least two forms, MAO A and MAO B, based on substrate selectivity, inhibition by various dmgs, and cloning experiments. Clorgyline [17780-72-2] is a specific inhibitor of MAO A, which displays a substrate specificity for NE and serotonin. Deprenyl [2323-36-6] is a selective inhibitor of MAO B, and displays a substrate preference for P-phenylethylamine and benzyl amine. Dopamine and tyramine are substrates for both enzymes. 

We now describe two applications of comparative modeling in more detail (1) Modeling of substrate specificity aided by a high accuracy model and (2) confinning a remote structural relationship based on a low accuracy model. 

JW Pitera, NR Munagala, CC Wang, PA Kollman. Understanding substrate specificity m human and parasite phosphoribosyltransferases through calculation and experiment. Biochemistry 38 10298-10306, 1999. 

Different side chains in the substrate specificity pocket confer preferential cleavage... 

The serine proteinases all have the same substrate, namely, polypeptide chains of proteins. However, different members of the family preferentially cleave polypeptide chains at sites adjacent to different amino acid residues. The structural basis for this preference lies in the side chains that line the substrate specificity pocket in the different enzymes. 

Engineered mutations in the substrate specificity pocket change the rate of catalysis... 

The Asp 189-Lys mutation in trypsin causes unexpected changes in substrate specificity... 

Asp 189 at the bottom of the substrate specificity pocket interacts with Lys and Arg side chains of the substrate, and this is the basis for the preferred cleavage sites of trypsin (see Figures 11.11 and 11.12). It is almost trivial to infer, from these observations, that a replacement of Asp 189 with Lys would produce a mutant that would prefer to cleave substrates adjacent to negatively charged residues, especially Asp. On a computer display, similar Asp-Lys interactions between enzyme and substrate can be modeled within the substrate specificity pocket but reversed compared with the wild-type enzyme. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Mutations in the specificity pocket of trypsin, designed to change the substrate preference of the enzyme, also have drastic effects on the catalytic rate. These mutants demonstrate that the substrate specificity of an enzyme and its catalytic rate enhancement are tightly linked to each other because both are affected by the difference in binding strength between the transition state of the substrate and its normal state. 

Craik, C.S., et al. Redesigning trypsin alteration of substrate specificity. Science 228 291-297, 1985. 

Graf, L., et al. Selective alteration of substrate specificity by replacement of aspartic acid 189 with lysine in the binding pocket of trypsin. Biochemistry 26 ... 

Wells, J.A., et al. Designing substrate specificity by protein engineering of electrostatic interactions. Proc. Natl. Acad. Sci. USA 84 1219-1223, 1987. 

The MYD analysis assumes that the atoms do not move as a result of the interaetion potential. The eonsequenees of this assumption have recently been examined by Quesnel and coworkers [50-55], who used molecular dynamic modeling techniques to simulate the adhesion and release of 2-dimensional particles from 2-D substrates. Specifically, both the Quesnel and MYD models assume that the atoms in the different materials interact via a Lennard-Jones potential

[Pg.153]

The definition is intended to differentiate these adhesives from merely sticky materials like flypaper or materials that may have only substrate specific adhesion. 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

The rearrangement has been found to be substrate specific. In some cases, the reaction proceeds as described above, i.e. using alkoxide in alcoholic solvent. In other cases, these conditions do not work well, or the reaction has been found to work better under pressure at elevated temperature in alcoholic solvents, in DMSO, DMF," or toluene. Rigorous exclusion of moisture and carbon dioxide is necessary."... 

For less activated substrates, specific tests are desirable. These are available in some cases. Thus Amstutz etal. showed that the reaction of 3-, 6-, and 8-bromoquinolines with piperidine at about 200° produced normal substitution products. 

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