Amide hydrolysis Trypsin

Acylation reaction, 171 Alanine, structure of, 110 Alcohol dehydrogenase, 205 Amide hydrolysis, see also Serine proteases Trypsin... 

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

A new, more general, way to combine ab initio quantum mechanical calculations with classical mechanical free-energy perturbation approach (QM/FE approach) to calculate the energetics of enzyme-catalysed reactions and the same reaction in solution has been reported." The calculated free energies were in fairly good agreement with the experimental data for the activation energies of the first test case, amide hydrolysis in trypsin and in aqueous solution. 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

Trypsin Ester, amide hydrolysis AOT/isooctane and CTAB/ chlaroform/isooct e [71]... 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

The substrate specificity of many esterases is not high (19) and the same is true of some proteases (amide-hydrolyzing enzymes), such as a-chymo-trypsin (12, 20). Amides may also serve as substrates for some esterases (21). Since esterases and proteases are widespread, hydrolysis of ester or amide linkages often accompanies other transformations by intact organisms. Soluble hydrolases are often present in supernatant fractions of mammalian microsomal preparations, and hydrolytic reactions may also occur when extracts of this type are used. Glycosidases, which catalyze the hydrolysis of... 

Modifications introduced by the mutations were central to the alteration of the specificities of the enzymes studied, which were capable of cleaving (3-casein at many new sites, for example, hydrolyzing the fragment Argl-Lysl05, reported to be a trypsin inhibitor (Bouhallab et al, 1997). Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the mutants of this type could be used for the hydrolysis of the lytically resistant protein structures. 

The trypsin family of serine proteases includes over 80 well-characterized enzymes having a minimum sequence homology of >21%. Two amino acid residues are absolutely conserved (Cysl82, Glyl96) within their active sites [26,27]. These proteases have similar catalytic mechanisms that lead to hydrolysis of ester and amide bonds. This occurs via an acyl transfer mechanism that utilizes proton donation by histidine to the newly formed alcohol or amine group, dissociation and formation of a covalent acyl-enzyme complex. 

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

Substrate specificity differences between boar acrosin and trypsin are not particularly manifest when using small substrates, but these enzymes show distinctly different kinetics of porcine ZP hydrolysis (34). The loss of 30% mass in the conversion from m - to m -acrosin has little effect on the kinetic analyses of inhibition and substrate preference with artificial substrates and small trypsin inhibitors, indicating that this excised portion of the enzyme contributes little to the topography of the active site (35). From Km analyses with amide and ester substrates of Arg and Lys, acrBSin prefers the Arg substrates over Lys, and Km differences between amide and ester substrates indicates that ac Ssin proceeds kinetically through a classical double displacement mechanism as does trypsin (36). 

Trypsin Hydrolysis of polypeptides, amides, and esters at bonds involving the carboxyl groups of L-arginine and l-lysine, yielding peptides of lower molecular weight. 

Trypsin was named more than 100 years ago. It and chymotrypsin were among the first enzymes to be crystallized, have their amino acid sequences determined, and have their three-dimensional structure outlined by x-ray diffraction. Furthermore, both enzymes hydrolyze not only proteins and peptides but a variety of synthetic esters, amides, and anhydrides whose hydrolysis rates can be measured conveniently, precisely and, in some instances, extremely rapidly. As a result, few enzymes have received more attention from those concerned with enzyme kinetics and reaction mechanisms. The techniques developed by the pioneers in these various fields have enabled other serine proteases to be characterized rapidly, and the literature on this group of enzymes has become immense. It might be concluded that knowledge of serine proteases is approaching completeness and that little remains but to fill in minor details. 

Note that the action of restriction endonucleases is analogous to the action of trypsin (Section 18.5B), which catalyzes the hydrolysis of amide bonds formed by tbe carboxyl groups of Lys and Arg and the action of chymotrypsin, which catalyzes the cleavage of amide bonds... 

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