Carboxypeptidase functional residues

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

The qualitative shape of the pH- cat/Am profile (Fig. 10 a) is typical of the profiles obtained for most carboxypeptidase A substrates 33). However, (as mentioned above), the quantitative aspects of these bell-shaped curves are highly dependent on substrate structure. Therefore, it is presently of little utility to attempt to correlate these profiles with the specific ionization of the various functional residues which are knowm to be present at the enzyme active site. 

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... 

Carboxypeptidase A"" (CPA, EC 3.4.17.1) is a proteolytic enzyme that cleaves C-terminal amino acid residues with hydrophobic side chains selectively. Several X-ray structures are available" The active site of CPA consists of a hydrophobic pocket (primary substrate recognition site) that is primarily responsible for the substrate specificity, a guanidinium moiety of Argl45 that forms hydrogen bonds to the carboxylate of the substrate, and Glu270, whose carboxylate plays a critical role, functioning either as a nucleophile to attack the scissUe carboxamide carbonyl carbon of the substrate or as a base to activate the zinc-bound water molecule, which in turn attacks the scissile peptide bond ". However, semiempirical calculations had shown that the direct attack of... 

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... 

By the use of a model system, Kimura et al. [17] tried to mimic the function of the two mechanistically most typical zinc(II) enzymes. Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate ion and its zinc(II) active site is bound to three histidine residues and a water molecule. Carboxypeptidase A (CPA, EC 3.4.17.1) catalyses the hydrolysis of the hydrophobic C-terminal amino acids from polypeptides, and its active-site zinc(II) is bound to two histidine residues, a glutamine residue and a water molecule which is hydrogen bound to a glutamine residue (Scheme 19). 

Fig. 1 Blocks of multiple sequence alignment of protein sequences of carboxypeptidases from B. taurus, Mus musculus, Rattus norvegicus, Neurospora crassa, Schizosaccharomyces pombe, Drosophila melanogaster, and Homo sapiens along with protein sequence from H. pylori (Uniprot accession code HPAG1 0372 from strain HPAG1). Numbers on the top correspond to amino acid residue number of the carboxypeptidase enzyme from B. taurus. Gray vertical columns indicate conserved residues. Amino acid residues corresponding to Glu-182 and His-306, which coordinate to zinc, are conserved, whereas another Zn-coordinating amino acid residue corresponding to His-179 is substituted by Gin in the Helicobacter sequence. Functionally important residues corresponding to Arg-237 are also conserved...

His-306 of carboxypeptidase from Bos taurusarc conserved. These amino acid residues are implicated in zinc coordination. Yet another functionally important residue that binds to substrate corresponding to Arg-237 of bovine enzyme is also conserved in the H. pylori sequence (Fig. 1) (30-32). 

The N-hydroxy amino acid derivatives are likely to be applicable to other metalloproteases. Thermolysin is inhibited irreversibly at pH 7.2 by ClCH2CO-DL-HOLeu-OCH3 where HOLeu is N-hydroxyleucine (47). The inhibition reaction involves coordination of the hydroxamic acid functional group to the active-site zinc atom of the enzyme. This then places the chloroacetyl group adjacent to Glu-143, an essential catalytic residue of thermolysin (see Figure 9). An ester linkage is formed and the enzyme is inactivated irreversibly. This reagent also inactivated two neutral metalloproteases from B. subtilis, but reacted only very slowly with carboxypeptidase A (t1/2 > 3 d). 

The three-dimendrmal structure of many enzyme molecules, including hydrolytic enzymes such as lysozyme, clymotiypsin, tibonudease, carboxypeptidase A, elastase, and papain, has been determined in recent years through the X-ray diffraction method, and the steric arrangement and function of amino add residues at the active site has been elucidated (i). 

Encron rizolipasc. endogenous pyrogen > interleukin-1. ENDOPEPTIDASE INHIBITORS act at one or other of the endopeptidase enzymes that cleave the C-terminal residue from oligopeptides or proteins (thus are stricdy proteinases). They can be divided into classes on the basis of their functional characteristics. These classes are dealt with separately in terms of their alternate names, notable substrates and inhibitors. They often act along with ectopeptidases - the carboxypeptidases and amino-peptidases. Endopeptidase inhibitors contain members of the metalloproteinase and serine protease families. Some are important neuropeptidases - concerned with degradation of... 

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