Bacterial carboxypeptidase

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. 

Carboxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11) is a bacterial enzyme not produced in mammalian cells. It has been used with promising success to target cytotoxic alkylating agents to tumor cells [60][61]. In a series of studies, a CPG2 monoclonal antibody con-... 

The site of action in the 3-lactam antibiotics is muramoylpentapeptide carboxypeptidase, an enzyme that is essential for cross-linking of bacterial cell walls. The antibiotic resembles the substrate of this enzyme (a peptide with the C-terminal sequence D-Ala-D-Ala) and is therefore reversibly bound in the active center. This brings the 3-lactam ring into proximity with an essential serine residue of the enzyme. Nucleophilic substitution then results in the formation of a stable covalent bond between the enzyme and the inhibitor, blocking the active center (see p. 96). In dividing bacteria, the loss of activity of the enzyme leads to the formation of unstable cell walls and eventually death. 

This enzyme [EC 3.4.16.4], also known as serine-type D-alanyl-D-alanine carboxypeptidase, catalyzes the hydrolysis of D-alanyl-D-alanine to yield two D-alanine. This enzyme comprises a group of membrane-bound, bacterial enzymes of the peptidase family Sll. They are distinct from the zinc D-alanyl-D-alanine carboxypeptidase [EC 3.4.17.14]. The enzyme also hydrolyzes the D-alanyl-D-alanine peptide bond in the polypeptide of the cell wall. In addition, the enzyme will also catalyze the transpeptidation of peptidyl-alanyl moieties that are A-acetyl-substituents of D-alanine. The protein is inhibited by j8-lactam antibiotics, which acylate the active-site seryl residue. 

The enzymatic colorimetric format is followed by the Penzyme test. This test is a qualitative enzymatic assay for rapid detection of -lactam residues in milk (28-30). The detection principle of the Penzyme test is based on measurement of the degree of inactivation of the enzyme oo-carboxypeptidase is involved in the synthesis of the bacterial cell wall by -lactam antibiotics. These residues bind specifically with the enzyme and inactivate it, thus interfering with bacterial cell wall formation. 

A bacterial peptidase splits a 20-residue fragment containing His 12 from the N-terminal end of RNase A. This "S-peptide" can be recombined with the rest of the molecule, which is inactive, to give a functional enzyme called ribonuclease S. In a similar way, residues 119-124 of RNase A can be removed by digestion with carboxypeptidase to give an inactive protein which lacks His 119. In this case, a synthetic peptide with the sequence of residues 111 -124 of RNase A forms a complex with the shortened enzyme restoring full activity.755... 

The action of p-lactam antibiotics is considered to be due to the formation of an acyl enzyme with carboxypeptidases and transpeptidases which are involved in the biosynthesis of bacterial cell walls38). A three-step mechanism involving a stable acyl-enzyme intermediate (El ), a participating active site serine residue, and a very slow decay process (k4.) was proposed [Eq. (9)]59). 

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation. 

The impetus for the newer work was the observation that benzyl clavulanate (11c) is a time-dependent inactivator of HLE (ICjq = 5 //M) whereas clavulanic acid (11b) is inactive [211, 212]. This finding led to the hypothesis that, since HLE is an endopeptidase whereas the bacterial serine proteinases are carboxypeptidases, quench(ing) the negative charge that the -lactam antibiotics normally require might yield HLE inhibitors. For synthetic reasons the group at Merck decided to use 7-aminocephalospo-ranic acids (1 Id) for most of their initial SAR studies. In contrast to the result with clavulanic acid, conversion of the cephalosporin 2 -carboxyl group in (1 Id) to an ester (He) was insufificent to transform the compound to an HLE inhibitor. 

Metalloprotease inhibitors - also known as metalloproteases or zinc proteases - are proteolytic enzymes of which the activity depends on metal ions, normally bound Zn -. Examples of metalloproteases are the pancreatic enzymes carboxypeptidase A and B, elastase, the well-characterized bacterial enzyme thermolysin and the collagenase family (found in both bacterial and mammalian cells, fibroblast collagenase, neutrophil elastase, gelatinase). 

Carboxypeptidase A Bovine, bacteria bacterial infection Hypertension Angiotensin 284-287... 

Clan SC contains peptidases with the a/P hydrolase fold bearing the catalytic triad in the order Ser, Asp, His. This clan includes the families (characteristic member in parentheses) S9 (prolyl oligopeptidase), S10 (carboxypeptidase C), S15 (Xaa-Pro dipeptidyl -peptidase), S28 (lysosomal Pro-Xaa carboxypeptidase), S33 (prolyl amino-peptidase), and S37 (Streptomyces PS-10 peptidase). The characteristic catalytic dyad Ser, Lys of dan SE is represented by the motif Ser-Xaa-Xbb-Lys, and the fold consists of helices and an a + P sandwich. The families of this clan Sll (penicillin-binding protein 5), S12 (Streptomyces R61 D-Ala-D-Ala carboxypeptidase), S13 (penicillinbinding protein 4) are involved in the biosynthesis, turnover and lysis of bacterial cell walls. 

The bacterial enzyme requires two divalent metal ions for activity, one playing an important role in the catalytic reaction. Zn Mn, Co Ni and Cd work in the binuclear metal center of the enzyme [709, 770]. On the basis of an analogy with the chemical mechanism for carboxypeptidase it appears that the metal acts as a Lewis acid for the polarization of the phosphorus-phosphoryl oxygen bond and also as a binding site for the hydrolytic water molecule. 

Of interest is the molecular mechanism by which the electron transfer takes place. For the bacterial camphor monooxygenase it was established by various independent methods that the putida redoxin forms a complex with the heme sulfur protein with a Km value of about 0.5-3 The carboxy terminal end of putida redoxin contains a tryptophan, which seems to participate in the binding, since its cleavage by carboxypeptidase A increases the Km for cytochrome P450 from 2-4 to 91 The formation of an enzyme-enzyme complex must also be postulated... 

Among many other peptide splitting enzymes such as (bacterial) subtilisin and thermolysin, (vegetable) papain, ficin and bromelain, (mammalian) cathepsin and others, the yeast enzyme carboxypeptidase Y finally deserves special mention. The enzyme is an exopeptidase, like carboxypeptidase A i.e. it catalyzes, rather unspecifically, the hydrolytic fission of the carboxy-terminal a-amino acids from a peptide chain. J.T. Johansen and his associates at the Carlsberg laboratory in Copenhagen showed about 10 years ago that CPD-Y is an effective catalyst of peptide bond synthesis [36]. 

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