And, endopeptidases

Proteins identified by their ability to bind labelled (3-lactam antibiotics in vivo and in vitro. The intrinsic activities of PBPs include transglycosylase/transpepti-dase, carboxypeptidase and endopeptidase activities required for the formation of the bacterial murein sacculus forming the bacterial cell wall. The enzymes are located in the cytoplasmic membrane. 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

The NC-IUBMB classifies peptidases (EC 3.4) into exopeptidases (EC 3.4.11-19), which remove one or a few amino acids, and endopeptidases (proteinases, EC 3.4.21-99), which catalyze the cleavage of peptide bonds away from either end of the polypeptide chain (Fig. 2.1). Exopeptidases are further subdivided into enzymes that carry out hydrolysis at the N-terminus or the C-terminus (Figs. 2.1 and 2.2). Thus, aminopeptidases (EC 3.4.11) cleave a single amino acid from the N-terminus [3] those removing a dipep-... 

MA 9 Families of exo- and endopeptidases (including several aminopeptidases in family Ml, and enkephalinase in family M13) Zn2+ Bound to //w-Glu-Xaa-Xaa-His (HEXXH motif) a/f ... 

The brush border enzymes in the intestine play a major role in peptide hydrolysis. Thus, both aminopeptidase and endopeptidase activities were detected with [Leu5]enkephalin as the substrate [146],... 

The in vitro hydrolysis of insulin has been shown to be catalyzed by exopeptidases and endopeptidases. Carboxypeptidase A (EC 3.4.17.1) cleaves the C-terminus of the B-chain (ThrB3°) and that of the A-chain (AsnA21) [145], Leucyl aminopeptidase (EC 3.4.11.1) cleaves the N-terminus of the B-chain (PheB1) and can continue to shorten it. But, leucyl aminopeptidase appears also able to cleave the N-terminus of the A-chain (GlyA1). In addition to these exopeptidases, entire insulin is also cleaved by endopeptidases of the... 

Disulfide Connectivities of a GM2 Activator Protein Fragment (24) Prepared by Digestion with Trypsin, S. aureus V8 Protease and Endopeptidase Asp-N [79 ... 

The metalloproteases include both exopeptidases (e.g., angiotensin-converting enzyme, aminopeptidase-M, and carboxypeptidase-A) and endopeptidases (e.g.,... 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

A protease hydrolyzes a peptide bond. Proteases have varying degrees of specificity, depending on the chemical nature of the R group and the location of the peptide linkage. Exopeptidases attack one or both ends of a polypeptide chain, and endopeptidases attack interior linkages. 

To improve the absorption of peptides and proteins, the employment of different enzymatic inhibitors has been suggested. For example, some authors have demonstrated the usefulness of inhibitors of both exo- and endopeptidases to promote calcitonin absorption across rat vaginal mucosa [93]. A direct relationship was found between the effect of peptidase inhibitors of in vitro degradation constant of calcitonin in vaginal mucosa homogenates and in vivo promotion of drug absorption, indicated by the decrease in plasma calcium levels after calcitonin administration in rats. The peptidase inhibitors tested were pepstatin, bestatin, and leupeptin. 

The nasal route of drug delivery avoids the liver first-pass effect, but the pseudo-first-pass effect owing to nasal metabolism of drugs is still a concern. Many enzymes such as carboxylesterase, aldehyde dehydrogenase, glutathione transferases, UDP-glucoronyl transferase, epoxide hydrolases, CYP-dependent monoxygenases, exo- and endopeptidases and proteases are present in the nasal mucosa.106 108,110,116 CYP enzymes are present abundantly in the olfactory epithelium.107,110... 

Proteases can be subdivided into two major groups exopeptidases cleaving the peptide bond proximal to the amino or carboxy terminal of the substrate, and endopeptidases cleaving distant from the termini (Rao et al., 1998). According to the functional group at the active site, proteases are further classified into four groups serine proteases, aspartyl proteases, cysteine proteases and metalloproteases. Based on the pH optimal for their functioning, proteolytic enzymes can be characterised as alkaline, neutral or acidic proteases. 

Figure 1.11 Generic peptide showing points of cleavage by exopeptidases and endopeptidases. Exopeptidases cleave...

Thus, rather than trial-and-error development of functionality, it should be possible to design functionality based on the principles of protein structure and function and the specificities of the enzymes used for modification. Use of immobilized exo- and endopeptidases in such technology could be especially attractive for the reasons listed in Table I, particularly since problems associated with autolysis would be eliminated and the extent of proteolytic reactions could be controlled with some precision. 

The distinction between exopeptidases and endopeptidases is merged for some members of the subfamily CIA. Dipep-tidylpeptidase I acts principally as an exopeptidase, removing N-terminal dipeptides, but may have some endopeptidase activity. Cathepsins B and H both possess endopeptidase activity but also possess exopeptidase activities. Cathepsin B acts as a peptidyl-dipeptidase, releasing C-terminal dipeptides. Cathepsin X is a carboxypeptidase. 

Protein metabolism is a key physiological process in all forms of life. Proteins are converted to amino acids and then catabolised. The complete hydrolysis of a polypeptide requires mixture of peptidases because individual peptidases do not cleave all peptide bonds. Both exopeptidases and endopeptidases are required for complete conversion of protein to amino acids. 

Proteins and peptides are accessible to enzymatic action due to the susceptibility of specific amino acid sequences, and such proteolysis is a naturally occurring metabolic process in vivo. Degradation pathways generally involve hydrolysis of peptide bonds by a variety of exopeptidases and endopeptidases, and the specific proteolytic enzymes associated with non-invasive routes of administration have been identified in some detail. Enzymatic activity varies depending on the delivery route and a qualitative rank ordering is shown in Table 1. Since a significant portion of dietary protein consumed by humans is assimilated by means... 

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