Thermolysin binding mode

The reversal of the peptidic functional groups is often used in peptide chemistry. The obtained retropeptides are generally more resistant to enzymatic attacks (Figure For thiorphan and rctro-thiorphan an identical binding mode to the zinc protease thermolysin was demonstrated. Similar inhibition values for thermolysin and neutral endopeptidase were observed, whereas, for another zinc protease, angiotensin-converting enzyme (ACE), noticeable differences for inhibition were found (Figure 15.47). 

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]. 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

The proteins which have been described fulfill a wide variety of physiological functions. Apparently there exist correspondingly large differences in their chemical structures and binding affinities. The mode of calcium binding is known without ambiguity for only parvalbumin and thermolysin. In these proteins oxygen coordination of calcium via... 

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. 

Holden, H.M., Tronrud, D ., Monzingo, A.F., Weaver, L.H. and Matthews, B.W. (1987) Slow- and Fast-Binding Inhibitors of Thermolysin Display Different Modes of Binding Crystallographic Analysis of Extended Phosphonamidate Transition-State Analogues, Biochemistry, 26, 8542-8553. 

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