Carboxypeptidase enzymatic mechanisms

Recently, molecular dynamics simulations of carboxypeptidase A have been employed to further our understanding of the enzymatic mechanism of carboxypeptidase A (Makinen et al. 1989 Banci et al. 1992 Banci et al. 1993 Stote and Karplus 1995). In an early study, molecular dynamics simulations were used to characterize the secondary structure motion of enzyme as well as the motion of the different subsites... 

Therefore, a key argument in regard to the proposed enzymatic mechanism of carboxypeptidase A is whether the carboxylate group of Glu-270 is steri-cally capable of participating efficiently in a nucleophilic reaction. In fact, such evidence has now been obtained by spectral characterization in the subzero temperature range (—60°C) study of a covalent acyl-enzyme intermediate obtained in the hydrolysis of the specific substrate 0-(trans-p-chlo-rocinnamoyl)-L-jS-phenyllactate by carboxypeptidase A (224). Furthermore, the results indicate that deacylation of the mixed anhydride intermediate is catalyzed by a Zn-bound hydroxide group. 

To elucidate the difference between the enzymatic and nonenzymatic participation of metal ions, it is clearly desirable to be able to compare the effect of a large number of metal ions upon the same reaction both in the presence and absence of the enzyme. For such a study to be feasible it is necessary to work with a metal-activated enzymatic reaction, which will also take place when the metal, but not the enzyme, is omitted. Such a reaction is the decarboxylation of oxaloacetic acid. The mechanism of metal catalysis of this reaction is similar to that assumed for carboxypeptidase, and can be represented as follows (44). 

Stable compounds which resemble the transition-state structure of a substrate in an enzymatic reaction are expected to behave as potent reversible inhibitors (1 ). Based on the X-ray crystallographic structure of the active site of carboxypeptidase A (CPA) (2), a mechanism was proposed in which a water molecule adds directly to the scissile carbonyl group of the substrate to give the tetrahedral intermediate 1, which collapses to products (3). We proposed to mimic this tetrahedral intermediate, similar to the transition state, with the stable tetrahedral phosphonic acid derivatives 2,... 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

It is well established that the same three-dimensional scaffolding in proteins often carries constellations of amino acids with diverse enzymatic functions. A classic example is the large family of a/jS, or TIM, barrel enzymes (Farber and Petsko, 1990 Lesk et ai, 1989). It appears that lipases are no exception to date five other hydrolases with similar overall tertiary folds have been identified. They are AChE from Torpedo calif arnica (Sussman et al., 1991) dienelactone hydrolase, a thiol hydrolase, from Pseudomonas sp. B13 (Pathak and Ollis, 1990 Pathak et al, 1991) haloalkane dehalogenase, with a hitherto unknown catalytic mechanism, from Xanthobacter autotrophicus (Franken et al, 1991) wheat serine carboxypeptidase II (Liao et al, 1992) and a cutinase from Fusa-rium solani (Martinez et al, 1992). Table I gives some selected physical and crystallographic data for these proteins. They all share a similar overall topology, described by Ollis et al (1992) as the a/jS hydrolase... 

The mechanism of T was proposed on the basis of X-ray crystallographic studies (65) on complexes (U) of carboxypeptidase A formed with phosphonate inhibitors and the linear correlation between log K, for the phosphonate inhibitors and logfccat/Xm for analogous amide substrates (66). The linear correlation was taken to suggest that the rate-determining transition state for the peptidase action would resemble U and that the enzymatic action proceeds through V. 

Bioaffinity chromatography is also a useful tool for the solution of the mechanism of enzymatic processes [140]. Akanuma et al. [141] employed this method for the study of the binding site of bovine carboxypeptidase B on the basis of a complex formation with immobilized substrate analogues of basic and aromatic amino acids. The problem of determining peptides of the active sites of enzymes or antibodies can be solved by the isolation of labelled specific peptides as is shown in section 4.7.5.7. Bioaffinity chromatography was also applied to the study of the molecular structures of human fibroblast and leucocyte interpherones [142]. 

The mechanism of the observed cellular action of lysinoalanine is not well understood (Finot et al., 1977, Finot, 1983 Engelsma et al., 1979 Reyniers, 1979 Leegwater and Tas, 1980). The possible interaction of LAL with metal ions needs to be explored, however, in view of the recent observations both here and by Hayashi (1982) that LAL inhibits the enzymatic activity of metallo-enzymes such as carboxypeptidase, which contains zinc as part of its active site. The inhibition appears reversible since carboxypeptidase activity was regenerated following the addition of zinc sulfate to the LAL-inactivated enzyme. Inhibition is not surprising since LAL contains three amino and two carboxyl groups and structurally resembles ethylenediaminetetraacetic acid (EDTA), a well-known metal chelator. 

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