Residues in carboxypeptidase

Modifications involving Glu-270 and Arg-145 have not given such positive results. However, modification of arginyl residues in carboxypeptidase by diacetyl results in loss of peptidase activity. 

Table VIII. Changes in Peptidase and Esterase Activities" on Modification of Functional Residues in Carboxypeptidase A...

Secondly, fluorescence emission spectra of proteins usually allow conclusions to be drawn about the environment of the main fluorescent groups present (mainly the L-tryptophanyl residues). Fluorescence, in addition to circular dichroism, indicated that L-tryptophanyl residues in carboxypeptidase Y were buried in an apolar unsymmetrical environment (c.d. also indicated 40% / -conformation). Similarly in L-alanine aminopeptidase and dihydrofolate reductase fluorescence suggested L-tryptophanyl residues located in hydrophobic surroundings. By contrast, in bacteriorhodopsin one or more Trp residues appear to be exposed to a polar medium. ... 

Bromoacetyl-p-aminobenzyl succinic acid alkylates a methionyl residue in carboxypeptidase B. As shown above, similar reagents modify amino acids other than methionyl residues, i.e., Tyr-248 and Glu-270. In those cases, the alkylating moiety was located in close proximity to the scissile bond of a normal substrate. Therefore, it would most likely interact with a residue that presumably functions in the catalytic step. The hydrophobic nature of bromoacetyl-p-aminobenzylsuccinate and the fact that the reactive side chain is carried on the aromatic part of the molecule suggests that the methionyl residue modified is part of the substrate recognition site in the hydrophobic pocket of carboxypeptidase B.i.0,16 This possibility is reminiscent of the role of Met-192 in chymo-trypsin, where it appears to function as a flexible hydrophobic lid on the substrate binding pocket. 

Phosphorescence and ODMR are additional spectroscopies that can be used to investigate intramolecular interactions that affect tyrosine residues in proteins and polypeptides/215,216) An example is tyrosine and tyrosinate in horse liver alcohol dehydrogenase.(202) The same approach has been used to study the role of tyrosine in the mechanism of action of carboxypeptidase B.(21/,218) jn botli these proteins, as in other proteins which contain both... 

This zinc-dependent enzyme [EC 3.4.15.1] (also known as dipeptidyl carboxypeptidase I, dipeptidyl-dipeptidase A, kininase II, peptidase P, and carboxycathepsin) catalyzes the release of a C-terminal dipeptide at a neutral pH. The enzyme will also act on bradykinin. The presence of prolyl residues in angiotensin I and in bradykinin results in only single dipeptides being released due to the activity of this enzyme, a protein which belongs to the peptidase M2 family. The enzyme is a glycoprotein, generally membrane-bound, that is chloride ion-dependent. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Another contrast between the zinc proteases and the carbonic an-hydrases concerns the zinc coordination polyhedron. The carbonic an-hydrases ligate zinc via three histidine residues, whereas the zinc proteases ligate the metal ion through two histidine residues and a glutamate (bidentate in carboxypeptidase A, unidentate in thermolysin). Hence, the fourth ligand on each catalytic zinc ion, a solvent molecule, experiences enhanced electrostatic polarization in carbonic anhydrase II relative to carboxypeptidase A. Indeed, the zinc-bound solvent of carbonic anhydrase II is actually the hydroxide anion [via a proton transfer step mediated by His-64 (for a review see Silverman and Lindskog, 1988)]. 

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. 

The catalytic glutamate residue (Glu-143) is located at the bottom of a narrow cleft, where it is bound to a water molecule and cannot approach the substrate as readily as can Glu-270 in carboxypeptidase. 

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. 

The 2.0 A electron density map of carboxypeptidase A shows three zinc-protein contacts (91). The ligands have been identified as histidine-69, glutamic acid-72 and histidine-196 (91, 101), where the numbers indicate the positions of the residues in the sequence counted from the N-terminal end. The geometry of the complex is irregular but resembles a distorted tetrahedron with an open position directed towards the active site pocket, and presumably occupied by water in the resting enzyme (91). The similarity with the tentative structure of the metal-binding site in carbonic anhydrase is striking. 

The first reported preparation of cross-linked enzyme crystals was by Quiocho and Richards in 1964 [1], They prepared crystals of carboxypeptidase-A and cross-linked them with glutaraldehyde. The material they prepared retained only about 5% of the activity of the soluble enzyme and showed a measurable increase in mechanical stability. The authors quite correctly predicted that cross-linked enzyme crystals, particularly ones of small size where the diffusion problem is not serious, may be useful as reagents which can be removed by sedimentation and filtration. Two years later the same authors reported a more detailed study of the enzymic behavior of CLCs of carboxypeptidase-A [2], In this study they reported that only the lysine residues in the protein were modified by the glutaraldehyde cross-linking. The CLCs were packed in a column for a flow-through assay and maintained activity after many uses over a period of 3 months. 

A peptide is incubated with the carboxypeptidase enzyme, and the appearance of free amino acids is monitored. In theory, the amino acid whose concentration increases first should be the C terminus, and the next amino acid to appear should be the second residue from the end. In practice, different amino acids are cleaved at different rates, making it difficult to determine amino acids past the C terminus and occasionally the second residue in the chain. 

The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxy-peptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group at the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963). 

It is a proteolytic enzyme, present in the intestine in its inactive form (zymogen), trypsinogen. Trypsinogen is converted into its active form, trypsin, by enteropeptidase, a specialized proteolytic enzyme secreted by intestinal cells. Some free trypsin formed also catalyses the conversion of trypsinogen into trypsin. Trypsin can also convert chymotrypsinogen and procarboxypeptidase into chymotrypsin and carboxypeptidase, respectively. Trypsin has different amino acid specificity when compared with other proteolytic enzymes. Trypsin hydrolyses those peptide bonds whose carboxyl groups are contributed by Lys or Arg residues and if the next residue is not proline. The number of smaller peptides resulting from trypsin action is equal to the total number of Arg and Lys residues in the protein plus one. 

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