Thermolysin zinc enzyme reactions

As was mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. These include both exopeptidases, like carboxypeptidases A and B, which remove amino acids from the carboxyl-terminus of proteins, albeit with different specificities, and endopeptidases, like thermolysin, which cleave peptide bonds in the interior of the polypeptide chain. They have almost identical active sites (Figure 12.5) with two His and one Glu ligands to the Zn +. It appears that the Glu residue can be bound either in a mono- or bidentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

It is primarily for the above reasons that, in the view of this author, it is not yet possible to rmequivocably define the mechanistic role played by zinc ion for any zinc-enzyme. Nevertheless, with the exception of thermolysin, it is possible to arrive at reasonable mechanistic hypotheses for the various zinc enzymes considered in this review through the examination of data derived from both kinetic studies and from studies at equilibrium through judicious application of the anthropomorphic approach to the description of reaction mechanisms (50). 

The observed normal isotope effect of 1.9 provides further evidence supporting the role of Asp55 as the general base. Namely, a normal isotope effect of 1.9 is most consistent with general base catalysis by an amino acid side chain, as inverse isotope effects are commonly observed when a zinc-bound water molecule, or hydroxide, is the attacking nucleophile. For example, the zinc-containing enzymes AMP deaminase [111], thermolysin [112], stromelysin [113], and a desuccinylase [114] are each believed to utilize a zinc-bound water as the nucleophile, and all of these reactions are characterized by an inverse deuterium isotope effect. This inverse isotope effect is thought to result from a dominant... 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

The N-hydroxy amino acid derivatives are likely to be applicable to other metalloproteases. Thermolysin is inhibited irreversibly at pH 7.2 by ClCH2CO-DL-HOLeu-OCH3 where HOLeu is N-hydroxyleucine (47). The inhibition reaction involves coordination of the hydroxamic acid functional group to the active-site zinc atom of the enzyme. This then places the chloroacetyl group adjacent to Glu-143, an essential catalytic residue of thermolysin (see Figure 9). An ester linkage is formed and the enzyme is inactivated irreversibly. This reagent also inactivated two neutral metalloproteases from B. subtilis, but reacted only very slowly with carboxypeptidase A (t1/2 > 3 d). 

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation. 

Leucine aminopeptidase is interesting in that its active site contains two zinc atoms which together bind and activate the water molecule [74]. Despite this enzyme containing a dinuclear metal center at its active site, its mechanism, and specifically its mode of proton transfers reactions, appear to follow the general theme established by thermolysin and carboxypeptidase Adenosine deaminase and other members of the family of nucleoside and nucleotide deaminases utilize zinc-bound water as the catalytic nucleophile to displace ammonia from the 6-position of purines or the 4-position of pyrimidines and in all cases display inverse solvent deuterium isotope effects ranging from 0.3 to 0.8 on fec/Kni [75-80]. These effects are reminiscent of those observed for metallopro-teases and have their origins, like those of the proteases, in fractionation factors for the protons of the bound water that are less than one. 

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