Thermolysin

Thermolysin is a metalloenzyme isolated from Bacillus thermoproteo-lyticus. It is a heat-stable extracellular endopeptidase of molecular weight 34,600. The enzyme catalyzes the hydrolysis of peptide bonds that have the amino group as part of hydrophobic residues such as phenylalanine, isoleucine, or leucine. 

Hydrolysis is fastest when both Rt and R, are hydrophobic residues. Latt et al. (4) showed that the isolated enzyme has one Zn2+ and four Ca2+. 

The Zn2+ is required for catalysis, while the Ca2+ ions are necessary for thermostability. 

This enzyme has been studied extensively by x-ray, kinetic, NMR, optical, circular dichroic, and fluorescence techniques. Thus, many approaches have been used to explore the role of the metal ions in catalysis and of other protein residues in substrate binding and catalysis. The review of this enzyme will serve to point out the information to be gained from using multiple biophysical approaches in understanding metalloenzyme catalysis. 

Thermolysin belongs to a class of proteases (called neutral proteases) which are distinct from the serine proteases, sulfhydryl proteases, metal-loexopeptidases, and acid proteases. Neutral proteases A and B from Bacillus subtilis resemble thermolysin in molecular weight, substrate specificity, amino acid content, and metal ion dependence. Since physiological substrates are most likely proteins, it is difficult to design simple experiments that can be interpreted in terms of substrate specificity and relative velocities. Therefore, studies of substrate specificity and other kinetic parameters must be carried out on di- and tripeptides so that details of the mechanism of catalysis can be obtained and interpreted simply. 

Thermolysin (TEN EC 3.4.24.28), a thermostable bacterial protease isolated from Bacillus thermoproteolyticus, has been studied as the prototype of zinc-metallopeptidases at a time where no crystal structure was available for this class of proteases [122]. Crystallographic analysis of a number of TLN/inhibitor complexes has allowed an understanding of the binding mode of these inhibitors and allowed the mechanism of action of this protease to be determined [122]. These seminal studies have greatly inspired the development of NEP inhibitors, given the close stmctural relationship between TEN and NEP [123]. To examine further the structural relationships between these two peptidases, various phosphinic peptides were prepared. One of these compounds (58, Table 1) exhibits a Ki value of 26 nM toward thermolysin and 22 nM toward NEP [124]. 

Endoplasmic reticulum (ER)-aminopeptidase (ERAP) O O II H II [X 6° ERAP-1 = 33 ERAP-2=11  

Inhibition of the zinc peptidase thermolysin has been the target of peptide mimetic effort by Bartlett and co-workers at the University of California at Berkeley. The researchers began by investigating the X-ray crystal structure of this enzyme with the known peptidic inhibitor Cbz-GlyP-Leu-Leu (Gly represents the phosphonic acid analogue of glycine). This inhibitor has a fCj of 9 nM against the enzyme. The authors reasoned that the inhibitor could be confer- 

The perturbation converting the NH to a CH2 was calculated in a similar manner and in advance of inhibitor synthesis. In this case, the AGaq and AGcom were -2.4 0.28 and -2.72 0.84 kcal/mol, respectively, which predicted a AAGbind to be -0.3 kcal/mol. This result is in close agreement with the experimental result of. -0.1 kcal/mol, determined after the prediction. Since, like the phosphonate ester, the phosphinate lacks the hydrogen bond to the carbonyl of Alai 13, it should be less potent than the phosphanamide inhibitor. However, it is more potent than the phosphonate ester due to more favorable desolvation (AGsoi = -2.4 0.3) and reduced electrostatic repulsion. 

These studies showed, for the first time, the usefulness of free energy simulation methods to understand enzyme-inhibitor interactions and demonstrated the predictive power of this method. Furthermore, these 

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G., Berendsen, H.J.C. The essential dynamics of thermolysin Confirmation of the hinge-bending motion and comparison of simulations in vacuum and water. Proteins 22 (1995) 45-54. 

Thermogravimetry Thermoid Thermolite Thermo luminescence Thermolysin... 

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... 

Although equihbrium-controUed peptide synthesis has been successfully used on a number of occasions, including thermolysin-catalyzed synthesis of aspartame (126) and semisynthesis of insulin (127), the method has a significant drawback a water-miscible organic cosolvent added to the reaction medium to suppress the ionization of unactivated carboxy components significantly reduces the reaction rate. 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Fig. 13. Relative sorption capacity of proteins by carboxylic CP Biocarb-T vs pH of solution 1) terrilytin, 2) insulin, 3) chymotrypsinogen, 4) pancreatic ribonuclease, 3) pepsin, 6) thymarine, 7) thermolysine, 8) haemoglobin, P) lysozyme. mma, — quantity of protein bonden on Biocarb-T by pHma (...

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... 

Tetrhedral intermediate, 172 Thermodynamic cycles, 186 Thermolysin, zinc as cofactor for, 204 Thrombin, 170 Torsional potential, 111 Transition states, 41-42,44, 45,46, 88, 90-92 in amide hydrolysis, 219-221 oxyanion hole and, 181 stabilization of, 181,181 carbonium ion, 154,155,156-161, 167-169 for gas-phase reactions, 43... 

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. 

Thermolysin acts simultaneously at several sites on the Ca -ATPase without accumulation of large fragments this property proved useful in the sequence analysis of the Ca -ATPase [78,79,82,83], and in the isolation of SH-group-containing peptides [257]. Small fragments also accumulate after treatment of sarcoplasmic reticulum with subtilisin [256]. 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

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. 

Figure 12 Catalytic mechanism of thermolysin and stromelysin-1. (A) The mechanism of thermolysin [54], (B) The mechanism of stromleysin-1 [10]. Equivalent residues to Tyr-157 and His-231 are not observed for stromelysin-1. The proposed mechanism for collagenase-1 [S3] is similar to stromelysin-1, but also involves Asn-180 (equivalent to Asn-162 in stromelysin-1). This residue cannot participate in stromelysin-1 due to an additional residue between Ala-165 and Asn-162. (Adapted from Ref. 10.)...

Mathews BW. Structural basis of the action of thermolysin and related zinc peptidases. Acc Chem Res 1988 21 333-340. 

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