Carboxypeptidases mechanism

Knowing how the protein chain is folded is a key ingredient m understanding the mechanism by which an enzyme catalyzes a reaction Take carboxypeptidase A for exam pie This enzyme catalyzes the hydrolysis of the peptide bond at the C terminus It is... 

FIGURE 27 19 Proposed mechanism of hydrolysis of a peptide catalyzed by carboxypeptidase A The peptide is bound at the active site by an ionic bond between its C terminal ammo acid and the positively charged side chain of arginine 145 Coordination of Zn to oxygen makes the carbon of the carbonyl group more positive and increases the rate of nucleophilic attack by water... 

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. 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Evidence against the covalent mechanism has been summarized by Mock, who has also proposed alternative general-base-catalyzed mechanisms for ther-molysin and carboxypeptidase A.143 He suggests that His-231 is the general base for thermolysin and the carboxy-terminal carboxylate for carboxypeptidase A. The one common feature of all the proposed mechanisms is the Zn2+ functioning as a Lewis acid to polarize the substrate. 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

Figure 12.5 Zinc-hydroxide reaction mechanism for peptide hydrolysis by carboxypeptidase A. (Reprinted with permission from Lipscomb and Strater, 1996. Copyright (1996) American Chemical Society.)...

Phosphorescence and ODMR are additional spectroscopies that can be used to investigate intramolecular interactions that affect tyrosine residues in proteins and polypeptides/215,216) An example is tyrosine and tyrosinate in horse liver alcohol dehydrogenase.(202) The same approach has been used to study the role of tyrosine in the mechanism of action of carboxypeptidase B.(21/,218) jn botli these proteins, as in other proteins which contain both... 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

A simplified representation of the postulated mechanism of carboxypeptidase is shown in Fig. 3.12 [2] [76]. This mechanism involves the following steps ... 

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). 

There are three fundamental questions concerning the mechanism of action of carboxypeptidase (1) How do metal ions affect ester and amide hydrolysis (2) Under what conditions will a neighbouring carboxyl group participate in ester and amide hydrolysis emd what is the mechanism of such participation (3) How will a metal ion affect... 

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. 

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). 

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

Fig. 33. The promoted-water tetrahedral intermediate (a) in the carboxypeptidase A mechanism is simulated by the binding of the ketomethylene substrate analog BBP (Fig. 32b) as the gm-diol(ate) (b). [Reprinted with permission from Christianson, D. W., Lipscomb, W. N. (1989) Acc. Chem. Res. 22,62-69. Copyright 1989 American Chemical Society.]...

---