Thermolysin-like proteases

The mechaiusm by which metalloproteinases execute catalysis has been of interest for many years. Most studies focused on carboxypeptidase A and thermolysin-like proteases for which extensive stmctural, chemical, and biochemical data are available. The first peptide hydrolysis mechanisms to be proposed... 

Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, van Den BB, Venema G, Ulbrich-Hofmann R, Eijsink VG (1997) Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. J Biol Chem 272 11152-11156 Matsuda A, Matsuyama K, Yamamoto K, Ichikawa S, Komatsu K (1987) Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J Bacteriol 169 5815-5820 Matsumura I, Wallingford JB, Surana NK, Vize PD, Ellington AD (1999) Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase. Nat Biotechnol 17 696-701... 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

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. 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

Standard mechanism inhibitors are classified strictly as inhibitors of serine proteases. There have been reports of inhibitors of other classes of proteases that have similar mechanisms to those of standard mechanism inhibitors, though. Initial studies on the streptomyces metalloprotease inhibitor (SMPl) suggest that it inhibits the metalloprotease thermolysin through a substrate-like binding mechanism (2). Similarly, staphostatin B, a cysteine protease inhibitor from Staphylococcus aureus, binds in a substrate-like manner in the active site of staphopain cysteine proteases. However, staphostatin B has a glycine PI residue, which adopts a backbone conformation that seems to prevent nucleophilic attack of the scissiie bond (3). 

For these thermodynamically controlled syntheses a variety of enzymes may be used, e.g. serine proteases like chymotrypsin and trypsin, cysteine proteases (thiol proteases) like papain, aspartate proteases like pepsin and metalloproteases like thermolysin. ... 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

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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