Carboxypeptidase kinetics

Frere JM et al (1975) Kinetics of interactions between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and (3-lactam antibiotics. Eur J Biochem 57 343-351... 

The kinetic constants for the carboxypeptidase A catalyzed hydrolysis at pH 9 and 25° were /ccal=61 s Km=0.29 mM. In other words, the enzyme afforded a rate enhancement of 11 orders of magnitude (kcJkchem= 4.7xlO11), and a catalytic proficiency of 15 orders of magnitude ((kL.JKm)/... 

From crystallographic studies, fast reaction kinetics and site-directed mutagenesis to produce mutant enzymes differing from the native enzyme in one or more specified residues, the mechanism in (46) has been proposed for the attack of water at the carbonyl carbon of benzoylglycylphenylalanine bound to carboxypeptidase (Christianson and Lipscomb, 1989). 

Subsequent to substrate binding, a promoted-water mechanism is favored for the hydrolysis of the scissile peptide linkage based on the results of chemical, kinetic, and structural investigations of carboxypeptidase A. A general mechanism is shown in Fig. 31, in which the zinc-bound water of the native enzyme is a nucleophile promoted both by zinc and by the general base Glu-270 (Christianson and Lipscomb, 1989). 

Gas-liquid chromatography used for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins was described in 1976 by Davy and Morris. Based on carboxypeptidase A digestion of the protein, the partially digested protein was removed and the amino acids released after known time intervals were analyzed by quantitative gas-liquid chromatography. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins. Thus the amino acid sequences were determined. 

An important difference between thermolysin and carboxypeptidase leads to the major uncertainty in the mechanism of carboxypeptidase. This difference is that the catalytic carboxylate of carboxypeptidase is far more sterically accessible. The crucial question is whether or not the carboxypeptidase-catalyzed hydrolysis of peptides proceeds via general-base catalysis, as in equation 16.26, or via nucleophilic catalysis, as in 16.27. Early kinetic work concentrated on establishing the participation of the various groups in catalysis. 

Although many of these general features are correct, these mechanistic conclusions are based upon the assumption that the properties of the enzyme in solution are conserved on crystallization. It appears from Raman studies on arsanilazotyrosine-248 carboxypeptidase that the enzyme exists in solution in a number of different forms.509 It is not certain that the form which crystallizes out is the kinetically active species. Indeed there is evidence that the kinetics of carboxypeptidase in solution differ from those of the enzyme crystals, with the crystalline enzyme being 1000-fold less active with some substrates.510 The interaction of Gly-L-tyr with Mn carboxypeptidase A, studied by 1H NMR techniques, does not involve coordination of the carboxyl group of the substrate, and may well represent a different conformation from the one studied by crystallographic techniques.511 Several conformational forms of the Cd11 carboxypeptidase A have been found in solution, while the enzyme exists in a different form in the crystal state.312... 

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. 

In the presence of a large excess of Co2+, both native (97) and cobalt (92) carboxypeptidase A show an approximately two-fold activity increase. The kinetics of the enzyme are very complex at moderate or high substrate concentrations and involve both apparent activation and inhibition by substrate (95). Under the standard assay conditions used in connection with the observed cobalt activation, all these complicating factors contribute significantly. The additional Co2+ possibly interferes with these secondary effects rather than being a participant in catalysis. Further experimentation is needed to clarify this detail. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Matrix-assisted laser desorption ionization is another ionization mode used for MS analysis. Enzymatically digested peptides have been studied using a 90-well microchip constmcted in a MALDI plate format (see Figure 7.41). Peptide digestion was initiated in the MALDI interface where the peptide hormone, adreno-corticotropin (ACTH) was mixed with the enzyme carboxypeptidase Y. The mixing process was self-activated in the vacuum conditions. Subsequent TOF MS analysis produced kinetic information of the peptide digestion reaction [820]. 

Table 15. Kinetic parameters of active and deglycosylated acid carboxypeptidases from Aspergillus saitoi toward Z-Glu-Tyr at pH 3.1 and 30°C...

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

Even though this dipeptide is turned over quite slowly, the complex examined is probably a non-productive one. Furthermore an analogous ester substrate has not been found, and it is known that carboxypeptidase behaves quite differently toward ester and peptide substrates. In particular, the kinetic parameters for peptide hydrolysis for a series of metal substituted carboxypeptidases indicate that fccat values can range from 6000 min for the cobalt enzyme down to 43 min for the cadmium enzyme 66). The values on the other hand are almost totally independent of the particular metal present. The exact opposite is true for ester hydrolysis. Km varies from 3300 M for the cobalt enzyme to 120 M for the cadmium enzyme while k<.at is essentially unchanged. 

Analyses of in situ DNA synthesis of Euglena gracilis identify zinc-dependent steps in the eukaryotic cell cycle and show that the derangements in RNA metabolism are critical determinants of the growth arrest associated with zinc deficiency. Combined use of microwave-induced emission spectrometry and micro gel emulsion chromatography shows the presence of stoichiometric amounts of zinc essential to the function of E. gracilis and yeast RNA polymerases, the reverse transcriptases" from avian myeloblastosis, murine leukemic and woolly type C viruses, and E. coli methionyl tRNA synthetase. These results stress the importance of zinc to both nucleic acid and protein metabolism. Transient-state kinetic studies of carboxypeptidase A show that zinc functions in the catalytic step of peptide hydrolysis and in the binding step of ester hydrolysis. 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

Carboxypeptidase A is one of the most intensely investigated zinc metalloenzymes. The enzyme as isolated contains 1 g-atom of zinc per protein 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 (46). Activity can be restored by readdition of zinc or one of a number of other di-valent metal ions (47). Through a combined use of chemical modification and transient state kinetic studies, it has been possible to determine the role of zinc in the catalysis of ester and peptide hydrolysis by this enzyme. 

The kinetics of action of carboxypeptidase are very complex. The pH-rate profile for CPA-catalyzed hydrolysis of peptides is bell shaped, with apparent pK values of about 6.5 and 7.5. The former pK value could be attributed to Glu-270. The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyl)pro-pionic acid leads to the establishment (with minimum complications from this relatively simple reaction) of a pK value of 6.03 for the Co" and Zn" CPA, which is probably due to Glu-270. The binding of the substrate to both Zn" and Co" CPA appears to depend on an enzyme-bound group with pXa = 7.56 and 8.29 respectively, and on a group with pXa>9. These are attributed to the ionization of Tyr-248 and the bound water molecule. 

Enke alins, hormones with morphine-like activities, have been synthesized with chymotrypsin and papain as well as carboxypeptidase Y under kinetically controlled conditions. ... 

Further commercial interest was found by the transpeptidation reaction of porcine insulin to human insulin, the latter only differing in the last amino acid of Ae B-chain (Ala-B30 to Thr-B30). This reaction proceeds under kinetic control with trypsin, carboxypeptidase Y or achromobacter lyticus protease I. ... 

If one examines the speed of reaction as measured by rate constants k and also the strength of the bonds formed, as reflected in the formation constants (A or P), one can find many instances where, for example, the zinc-containing metalloprotein carboxypeptidase would have been kinetically and thermodynamically better placed to undertake this role in vivo had the zinc have been replaced with cobalt(n). Similarly, there are kinetic and thermodynamic reasons for replacing the ferrous ion in porphyrins with cobalt(ii) or with copper ions. 

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