Carboxypeptidase models

Breslow, R. and Mcallister, C, Intramolecular bifunctional catalysis of ester hydrolysis by metal ion and carboxylate in a carboxypeptidase model, J. Am. Chem. Soc., 1971, 93, 7096-7097. 

Schepartz, A. and Breslow, R., Hydrolysis of an amide in a carboxypeptidase model using CoIII and bifimc-tional catalysts, /. Am. Chem. Soc., 1987,109,1814-1826. 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

Micelles in water are described as spherical aggregates of a surfactant monomer27 30). They somewhat resemble to enzyme proteins in structures and functions, although the details are yet the subjects of recent controversies 29,30). There are numerous studies of micellar models of enzymes 28), but the examples of those of metalloenzymes are very few 31 37). In particular, there are no examples of micellar models of carboxypeptidase or carbonic anhydrase except ours 36,37). 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Aloy, P., Companys, V., Vendrell, J. et al. The crystal structure of the inhibitor-complexed carboxypeptidase D domain II and the modeling of regulatory carboxypepti-dases. /. Biol. Chem. 276 16177-16184,2001. 

The goal of the experiments we report was to create new structural model complexes for gluzincins or carboxypeptidases. With [Zn (bdtbpza)Cl] (12) for the first time a tetrahedral zinc complex with a monoanionic W,W,0-tridentate using a carboxylate 0-donor was synthesized (41). A comparison of the molecular structure of 12 with the coordination environment of the enzymes indicates its significance... 

The potential of the MALDI-MS-based assay scheme for the quantification of low molecular weight products and substrates directly from reaction mixtures has been described by Bungert et al. [8]. The glucose oxidase-based conversion of glucose to gluconolactone and the carboxypeptidase A-mediated cleavage of hippuryl-L-phenylalanine were chosen as model systems (Fig. 8.4). 

The three hydrolytic enzymes that have been discussed, a-chymo-trypsin, carboxypeptidase A, and lysozyme, cover a wide range of substrate types and mechanistic possibilities. Formulation of principles which might apply to enzymatic catalysis in general is difficult from such a small sampling, but certain features of the enzymatic and model reactions warrant some comment. 

Zinc may function to promote the nucleophilicity of a bound solvent molecule in both small-molecule and protein systems. The p/Ca of metal-free H2O is 15.7, and the p/Ca of hexaaquo-zinc, Zn (OH2)6. is about 10 (Woolley, 1975) (Table III). In a novel small-molecule complex the coordination of H2O to a four-coordinate zinc ion reduces the to about 7 (Groves and Olson, 1985) (Fig. 2). This example is particularly noteworthy since it has a zinc-bound solvent molecule sterically constrained to attack a nearby amide carbonyl group as such, it provides a model for the carboxypeptidase A mechanism (see Section IV,B). To be sure, the zinc ligands play an important role in modulating the chemical function of the metal ion in biological systems and their mimics. 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

Wang, X., Smith, P. L., Hsu, M. -Y, Ogletree, M. L., Schumacher, W. A. (2006). Murine model of ferric chloride-induced vena cava thrombosis evidence for effect of potato carboxypeptidase inhibitor. J. Thromb. Haemost., 4, 403 10. 

Model L The Use of Synthetic Y Zeolites as Models for the Migration of Zn2+ Ions in the Enzyme Carboxypeptidase A... 

We shall now briefly outline some of the features of the zinc metalloenzymes which have attracted most research effort several reviews are available, these are indicated under the particular enzyme, and for more detailed information the reader is referred to these. Attention is focussed here, albeit briefly, on carbonic anhydrases,1241,1262,1268 carboxypeptidases, leucine amino peptidase,1241,1262 alkaline phosphatases and the RNA and DNA polymerases.1241,1262,1462 Finally, we examine alcohol dehydrogenases in rather more detail to illustrate the use of the many elegant techniques now available. These enzymes have also attracted much effort from modellers of the enzymic reaction and such studies, which reveal much interesting coordination chemistry and often new catalytic properties in their own right—and often little about the enzyme system itself (except to indicate possibilities), will be mentioned in the next section of this chapter. 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Eiichi Kimura is retired from the Department of Medicinal Chemistry at Hiroshima University in Japan. His recent research interests have included the supramolecular chemistry of macrocyclic polyamines and their use in molecular recognition and as zinc-enzyme models. These interests have led to the development of fluorophore sensors for Zn(II) [8] use of macrocycles to effect selective recognition of anions [9], nucleobases in polynucleotides [10], thymidine mono- and diphosphate nucleotides (11), carbonic anhydrase and carboxypeptidase [12], and development of Zn(II)-macrocycle anti-HIV agents [13], In May 2004, he received a Purple Ribbon Award from the Emperor of Japan. 

Kimura, E. (2001) Model studies for molecular recognition of carbonic anhydrase and carboxypeptidase,... 

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). 

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