Thermolysin active-site structure

Fig. 2. Thermolysin active site. The structure shown is of a substrate bound to the active site 2n2+ and amino acid residues. From Kessler and Matthews (JO). Reprinted with permission of The American Chemical Society.

VanX is a carboxypeptidase that hydrolyzes the amide bond of a dipeptide, D-alanyl-D-alanine (D-Ala-D-Ala). This enzyme is essential for vancomycin-resistant enterococci (VRE), because it produces peptidoglycan precursors terminating in D-alanyl-D-lactate (D-Ala-D-Lac) in place of D-Ala-D-Ala, resulting in a 100-fold decrease in the affinity with vancomycin.Scheme 5 depicts a proposed mechanism of VanX, where the active-site structure is similar to those of CPDA and thermolysin. The Zn -bound H2O activated by Glul81 attacks... 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Fig. 3. Structure of the inhibitor phosphoramidon. The tetrahedral phosphorus atom binds at the active site of thermolysin and mimics the transition-state complex.

Holmes, M. A., D. E. Tronrud, and B. W. Matthews, Structural analysis of the inhibition of thermolysin by an active-site-directed irreversible inhibitor. Biochem. 22 236, 1983. 

Thus, many metal ions catalyze the hydrolysis of esters [7,8], amides [9], and nitriles [10] via electrophilic activation of the C=0 or C=N group. This type of catalysis is characteristic of coordination complexes and is very common in metalloenzyme-mediated processes. Zinc(II), for example, is a key structural component of more than 300 enzymes, in which its primary function is to act as a Lewis acid (see Chapter 4). The mechanism of action of zinc proteases, e.g., thermolysin, involves electrophilic activation of an amide carbonyl group by coordination to zinc(II) in the active site (Figure 4). 

Zinc proteinases contain a tightly-bound active site Zn2 + ion and a carboxylate group in the two zinc proteinases whose X-ray crystal structures are known, carboxypeptidase A and thermolysin, these are Glu-270 and Glu-143 (Lipscomb, 1983). One thus has at least the following three possibilities for the initial catalytic event ... 

Protein structure determinations have identified several examples of one domain inserted within another. One example is the E. coli DsbA protein, which catalyzes the formation of disulfide bonds in the periplasm. The enzyme consists of two domains a thioredoxin-like domain that contains the active site, and an inserted helical domain similar to the C-terminal domain of thermolysins (Martin et al., 1993). The inserted domain forms a cap over the active site, suggesting that it plays a role in binding to partially folded polypeptide chains before oxidation of... 

Gluzincins are proteins with the consensus HexxH( > 20)E, where the third zinc ligand, a glutamate, lies at least 20 residues C-terminal to the zincin motif (Table 2). The archetypical protein of this class, thermolysin consists of two domains, with the active site located between the N-terminal catalytic domain and the all alpha C-terminal domain (Fig. 7). With the limited data available, a comparison of topological matches of the observed structures with their cor-... 

Figure 7. The structure of thermolysin. Ribbon representation of the structure of thermolysin (silver, Brookhaven Databank [53] code 3TLN) shown with a bound inhibitor (green). The catalytic zinc atom (cyan) and structural calcium atoms (magenta) are shown. The active site is located between the N-terminal zinc protease domain and the alpha helical C-terminal domain. Zinc binding residues are in blue and the residue assisting catalysis is shown in red.

Z. R. Wasserman and C. N. Hodge. Fitting an inhibitor into the active site of thermolysin A molecular dynamics case study. PROTEINS Structure, Function and Genetics, 24 227-237, 1996. 

A structurally similar active site to that of carboxypeptidase A is found for the endopeptidase thermolysin.77 While several crystallographic and biochemical studies favor a zinc hydroxide mechanism for thermolysin (involving Glu-143 as a general base),80 in an alternative proposed mechanism for this enzyme, the zinc center is proposed to activate the substrate for nucleophilic attack by a non-coordinated water molecule (Scheme 14).81,82... 

The sections below deal with the biochemistry of the Ca " " ion only insofar as it is involved in catalysis (i.e. enzymatic activity.) They will not deal with Ca " bound to enzymes in which this ion plays a purely passive structural role as in the case of the Zn " " endoproteinase thermolysin which binds a Zn " " ion at the active site and four Ca ions elsewhere which stabilize the structure . Removal of the Ca " " causes this enzyme to autolyze (self-digest) and become inactive, but Ca is not directly involved in the catalytic function. Many examples of Ca acting in a passive structural rule are known. ... 

In the C-terminal domain are five helices in a closed bundle. This characteristic fold is typical of thermolysin-like peptidases. Clan MC contains metallocarbox-ypeptidases which belong to only one family (M14) which is divided into the subfamilies A, B and C. Typical for this clan is that one zinc ion is tetrahedrally coordinated by a water molecule, two histidine and one glutamate residues. Clan MF includes aminopeptidases that require cocatalytic zinc ions for their enzymatic activity. The well-known leucyl aminopeptidase has a two-domain structure bearing the active site in the C-terminal domain. Whereas exopeptidases of clan MG require cocatalytic ions of cobalt or manganese, clan MH contains the third group of metallopeptidases that also require cocatalytic metal ions, but here these are all zinc ions. The third clan in which cocatalytic metal ions are necessary is clan MF with zinc or manganese. Only one catalytic zinc ion is required for peptidases of clans MA, MB, MC, MD and ME. 

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