Thermolysin active site

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.

W. C. Guida, R. S. Bohacek, and M. D. Erion,/. Comput. Chem., 13,214 (1992). Probing the Conformational Space Available to Inhibitors in the Thermolysin Active Site Using Monte Carlo/Energy Minimization Techniques. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

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... 

DePriest SA, Mayer D, Naylor CB, Marshall GR. 3D-QSAR of angiotensinconverting enzyme and thermolysin inhibitors a comparison of CoMFA models based on deduced and experimentally determined active site geometries. J Am Chem Soc 1993 115 5372-84. 

Figure 1. Ribbon diagram of thermolysin complexed the inhibitor Cbz-GlyP-NH-Leu-Leu (stick) and Zn (sphere) in the active site (PDB code 5TMN).

Figure 12.4 Active sites of thermolysin and carboxypeptidases A and B. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

Fig. 11. (a) Active site of phenylalanine hydroxylase PheOH (PDB-Code IDMW) (97) and (b) active site Zentrum of thermolysin (THL) (PDB-Code ITHL) (98). 

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. 30. Important active-site residues of carboxypeptidase A (CPA) and thermolysin (TLN) and a general scheme for the active sites of related zinc proteases.

In both carboxypeptidase A and thermolysin the active site Zn2+ is chelated by two imidazole groups and a glutamate side chain (Fig. 12-16). In carboxypeptidase A, Arg 145, Tyr 248, and perhaps Arg 127 form hydrogen bonds to the substrate. A water molecule is also bound to the Zn2+ ion. The presence of the positively charged side chain of Arg 145 and of a hydro-phobic pocket accounts for the preference of the enzyme for C-terminal amino acids with bulky, nonpolar side chains. The Zn2+ in thermolysin is also bound to two imidazole groups and that in D-alanyl-D-alanyl carboxypeptidase to three. 

An 80- to 90-residue N-terminal propeptide domain contains a cysteine whose -S group binds to the active site zinc, screening it from potential substrates. The central catalytic domain is followed by a hinge region and a C-terminal domain that resembles the serum iron binding and transporting hemopexin.427/436 The mechanism of action is probably similar to that of thermolysin.430... 

A metalloenzyme peptide deformylase removes the formyl groups from the N termini of bacterial proteins. Although the active site is similar to that of thermolysin,443 the Zn2+ form of peptide deformylase is unstable. Both Ni2+ and Fe2+ form active, stable... 

The pancreatic carboxypeptidases are not homologous with thermolysin (and the G protease), but their active sites have evolved convergently to having striking similarities. 

The preceding study is directly applicable to understanding the active site of thermolysin, since a recent kinetic study (6) comparing neutral protease from B. subtilis with thermolysin showed that the two enzymes have identical pH rate profiles that peak near pH 7. 

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

A novel approach for determining metal-metal distances in proteins in solution was applied to thermolysin by Horrocks et al. (16), As mentioned earlier, thermolysin has four Ca2+ binding sites. Three of the four can be replaced by trivalent lanthanide ions. Tb3 + fluorescence is enhanced when Tb3+ is bound to the so-called 1 Ca2+ site of thermolysin. When Co2+ is added to the Zn2 + active site, the fluorescence of bound Tb3+ is quenched by... 

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