Carboxypeptidase, esterase activity specificity

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 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. 

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. 

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. 

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