Carboxypeptidase, esterase activity

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 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. 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

Acetylation and iodination of discrete tyrosyl residues have been achieved, leading to an abolishment of peptidase activity and an enhancement of esterase activity for both native and cobalt carboxypeptidase A (100). 

A metal atom is essential to the catalytic activity of carboxypeptidase A (53). The enzyme, as isolated, contains one gram atom of zinc per molecular weight of 34,600. Removal of the metal atom, either by dialysis at low pH or by treatment with chelating agents, gives a totally inactive apoenzyme. Activity can be restored by readdition of zinc or a number of other divalent metal ions (Table VII). The dual activity of carboxypeptidase towards peptides and esters is quite sensitive to the particular activating metal ion. Thus, the cobalt enzyme has twice the activity of the native zinc enzyme toward peptides but the same activity toward esters. Characteristic peptidase and esterase activities are also observed for the and Mn enzymes as well while the Cd ", Rh ", and Pb " en-... 

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

Table IX. Differences in Mechanistic Aspects of Peptidase and Esterase Activities of Carboxypeptidase A...

Figure 9, Mechanistic schemes for the peptidase and esterase activities of carboxypeptidase A...

Enzyme-catalyzed hydrolysis, exploiting the esterase activity of proteases such as trypsin and chymotrypsint ° l or carboxypeptidase has opened alternative routes to the deprotection of several peptide methyl, ethyl, and ferf-butyl esters. In fact, methyl, ethyl, and benzyl esters are successfully hydrolyzed from protected peptides using the alkaline protease from Bacillus subtilis or alcalase from Bacillus licheniformis which accepts... 

Carboxypeptidase A has esterase activity as well as peptidase activity. In other words, the compound can hydrolyze ester bonds as well as peptide bonds. When carboxypeptidase A hydrolyzes ester bonds, Glu 270 acts as a nucleophilic catalyst instead of a general-base catalyst. Propose a mechanism for the carboxypeptidase A-catalyzed hydrolysis of an ester bond. 

More recently, Kang and Storm 171) have shown that conversion of cobalt(II) carboxypeptidase A to the corresponding cobalt (III) enzyme by hydrogen peroxide oxidation is accompanied by the complete loss of peptidase activity when assayed with CbzGly-L-Phe. Nevertheless, the esterase activity, as measured by the rate of hydrolysis of 0-(N-benzoyl-Gly)-D,L-phenyllactic acid, remains unaffected by this transformation. Cobalt(III) complexes are characterized by extremely slow exchange rates due to the transformation from a d (Coll) to a... 

In my own laboratory at Tel-Aviv University we have been testing the effects of different monoclonal antibodies on the activity of carboxypeptidase A [31]. We have found some monoclonal antibodies that inhibit the esterase and peptidase activities of the enzyme, some that inhibit the esterase but not the peptidase activity, others that block the peptidase but not the esterase activity, and yet others that bind to the enzyme but do not markedly affect either of its catalytic activities. Some of the monoclonal antibodies seem to modify the conformation of carboxypeptidase A, thus greatly altering the kinetic parameters of the enzyme. 

Trypsin, chymotrypsin, and carboxypeptidase, long thought to be specific only for the hydrolysis of peptide bonds also display a specific esterase activity. 2 Trypsin, for example, splits ammonia from benzoylargininamide and as well splits ethanol from benzoylarginine ethyl ester. No activity, however, is shown toward ethyl butyrate, a substrate for an aliphatic esterase. 

Table 2.2.3.3 Determination of the carboxypeptidase and esterase side-activities of Si HNL in comparison with carboxypeptidase II from wheat. ...

Clan SC peptidases are a/p hydrolase-fold enzymes that consist of parallel P-strands surrounded by a-helices. The a/p hydrolase-fold provides a versatile catalytic platform that, in addition to achieving proteolytic activity, can either act as an esterase, lipase, dehalogenase, haloperoxidase, lyase, or epoxide hydrolase (18). Six phylogenetically distinct families of clan SC are known, and oifly four of them have known structure. Catalytic amenability of the a/p hydrolase-fold may underlie why clan SC peptidases are the second largest family of serine peptidases in the human genome. Other mechanistic classes need not use the catalytic serine and instead use cysteine or glutamic acid (19). Clan SC peptidases present an identical geometry to the catalytic triad observed in clans PA and SB, yet this constellation is ordered differently in the polypeptide sequence. Substrate selectivity develops from the a-helices that surround the central P-sheet core. Within clan SC, carboxypeptidases from family SIO are unique for their ability to maintain... 

Yet another example of the catalytic triad has been found in carboxypeptidase II from wheat. The structure of this enzyme is not significantly similar to either chymotrypsin or subtilisin (Figure 9.15). This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. These enzymes all make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. 

The substitution of another metal for that present in the native state or the removal of any metal is the simplest chemical modification for a metalloenzyme. Marked changes in activity are usually observed in either case. Substitution of cadmium for zinc first demonstrated a difference in the esterase and peptidase activities of carboxypeptidase A (47). The activity of [(CPD)Cd] toward Bz-Gly-L-OPhe is increased, but that enzyme is virtually inactive toward Cbz-Gly-L-Phe. 

In this superfamily, esterases, oxidoreductases, dehalogenases and carboxypeptidases can also be found [73 and references therein]. Characteristically, these enzymes consist of a highly conserved -sheet region, surrounded by ct-helical domains. A variable so called cap region completes their structural appearance. The active site of Hevea oxynitrilase has been found to lie deep inside the protein molecule and is accessible via a narrow tuimel [73] (Fig. 4). 

The DhiA enzyme functions as a monomer ( 35 kDa) and is composed of two domains a main domain and a cap domain (Figure 2(a)). The main domain consists of a mostly parallel eight-stranded /3-sheet connected by ct-helices on both sides of the sheet. The cap domain is composed of five ct-helices with intervening loops. The active site is an occluded hydrophobic cavity located at the interface of the two domains. The overall fold of the main domain is the hallmark of the o //3-hydrolase fold superfamily of enzymes, to which lipases, esterases, carboxypeptidases, and acetylcholinesterases also belong. These superfamily members catalyze the hydrolysis of ester and amide bonds via a two-step nucleophilic substitution mechanism similar to that of serine proteases. 

Carboxypeptidase A (approx. 35 kDa MW, Sigma Chemical) functions both as a peptidase and an esterase it is in this latter mode that it can serve as a detector for cholinesterase inhibitors. Unlike the other enzymes such as AChE or BChE, it does not have a serine residue in the active site. TPPSi forms a complex with the enzyme and, upon challenge with the cholinesterase inhibitor eserine (physostigmine) in water, exhibits a change in the absorbance spectrum with a new peak and a marked increase in absorbance at 423 nm. This suggests TPPS, may not be completely displaced from the active site. For actual sensor operations, the use of an enzyme such as BChE or carboxypeptidase in place of (or in addition to) AChE will allow for potential identification of the analyte based on different specificities/sensitivities of the enzyme. Enzymes such as OPH, which are not readily available, may be difficult to obtain in large quantities the supply of AChE is often limited perhaps due to the capture of electric eels, while proteins such as BChE (from horse blood) and carboxypeptidase (pancreas) are more readily available from slaughterhouses. 

Pancreatic enzymes a group of at least 12 digestive enzymes, including some of the most investigated of all enzymes Autolysis of the pancreas does not occur, because the proteolytic enzymes, trypsin, chymo-trypsin A and B, elastase and carboxypeptidase A and B, and phospholipase A2 are synthesized and stored in the pancreas as inactive zymogens. The other P. e. require effectors for optimal activity, which are present in the duodenum. Trypsin inhibitors in the pancreatic tissue and secretion afford additional protection against proteolytic destruction by active P.e. With the exception of cholesterol esterase (M, 400,000), the M, of P.e. lie between 13,700 (ribonu-clease) and 50,000 (a-amylase). 

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