Specificity substrate

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. 

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). 

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." 

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

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. 

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

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. 

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  

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

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. 

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

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

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. 

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. 

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. 

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 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. 

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