Peptidase , active site studies

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

The role of certain residues in the enzyme mechanism has been confirmed by chemical modification studies, notably for tyrosine. 14 Modification of tyrosyl residues (for example acetylation or nitration) leads to loss of peptidase activity and enhancement of esterase activity. The presence of the inhibitor -phenylpropionate protects two tyrosine residues from acetylation. Those are Tyr-248 and probably Tyr-198, which is also in the general area of the active site. The modified apoenzyme has lower affinity for dipeptides, as might be expected from the loss of hydrogen bonding between Tyr-248 and the peptide NH group. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

An understanding of the molecular interactions between the acylenzyme and the attacking nucleophilic amine component allows an optimization of the acyl transfer efficiency. The efficiency of the nucleophilic attack of the amine component depends essentially on an optimal binding within the active site by S - P interactions (Fig. 12.5-11). Consequently, more information on the specificity of the S subsites of serine and cysteine peptidases are useful, which can be obtained by systematic acyl transfer studies using libraries of nucleophilic amine components. According to the definition of the p value (see above) small values of p indicate high S subsite specificity for the appropriate amine component in peptidase-catalyzed acyl transfer reactions. 

Serine peptidases, serine proteases the most studied class of peptidases. They have a reactive serine residue, for example, the hydrolysis of a peptide substrate involves an acyl enzyme intermediate in which the hydroxyl group of Ser (chymotrypsin numbering system) is acylated by the acyl moiety of the substrate, thus releasing the amine fragment of the substrate as the first product. The formation of the acyl enzyme is the slow step in peptide bond hydrolysis, but acylenzyme often accumulates in the hydrolysis of ester substrates. The acyl enzyme thus formed will be the same for a series of substrates which differ in their leaving group. The active site of serine peptidases is complementary in stmc-ture to the transition state of the reaction, a structure which is very close to the tetrahedral adduct of Ser and the carbonyl... 

Cyclobutanones are usually formed by the classical ketene-olefin [2 H- 2] cycloaddition method. Compound 77, which is related to the penam family, was readily obtained [55, 56] from ethyl 2,3-dihydrothiophene-3-carboxylate and dichloroketene (Scheme 28). The binding of this compound to the active site of R61 D,D-peptidase was proved by an X-ray crystallographic study [57]. 

The depsipeptides 321, [182-185] which are ester analogs of acyl-o-alanyl-o-alanine peptides, were found to be as susceptible to attack by hydroxide as the penicillins. Although simple o-alanyl-o-alanine peptides are not substrates of typical p-lactamases, the depsipetides are. They are also substrates of penicillin-sensitive D,D-carboxypeptidases. Depsipeptides are therefore excellent probes in the comparative studies of the active sites of p-lactamases and D,o-peptidases. 

Nabeno M, Akahoshi F, Kishida H, Miyaguchi I, Tanaka Y, Ishii S, Kadowaki T (2013) A comparative study of the binding modes of recently launched dipeptidyl peptidase IV inhibitors in the active site. Biochem Biophys Res Commun 434 191-196 Thoma R, Loffler B, Stihle M, Huber W, Ruf A, Hennig M (2003) Structural basis of pro-line-specific exopeptidase activity as observed in human dipeptidyl peptidase-IV. Structure 11 947-959... 

Enzymological studies using various group-specific inactivators of proteases indicated that renin belongs to the class of acidic proteases. The structural requirements of renin substrates were studied to elucidate the exceptionally selective substrate specificity of this peptidase whose only known function is to generate angiotensin I. These studies have led to the design and synthesis of active site-directed inhibitors of renin. 

The carboxypeptidases are released from their inactive precursors in the pancreatic juice of animals. The most studied example is bovine carboxypeptidase A, which contains one mole of zinc per protein molecular weight of 34 500. These enzymes cleave the C-terminal amino acid residue from peptides and proteins, when the side-chain of the C-terminal residue is aromatic or branched aliphatic of l configuration. At least the first five residues in the substrate affect the activity of the enzyme. The enzyme also shows esterase activity. Esters and peptides inhibit each other competitively, indicating that the peptidase and esterase sites overlap, even if they are not the same. 

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