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The selectivehydrolysis of a peptide or amide bond (−(O)C–NH−)by a synthetic metallopeptidase is required in a wide range of biological,biotechnological, and industrial applications. In nature, highly specializedenzymes known as proteases and peptidases are used to accomplish thisdaunting task. Currently, many peptide bond cleaving enzymes and syntheticreagents have been utilized to achieve efficient peptide hydrolysis.However, they possess some serious limitations. To overcome theseinadequacies, a variety of metal complexes have been developed thatmimic the activities of natural enzymes (metallopeptidases). However,in comparison to metallopeptidases, the hydrolytic reactions facilitatedby their existing synthetic analogues are considerably slower andoccur with lower catalytic turnover. This could be due to the followingreasons: (1) they lack chemical properties of amino acid residuesfound within enzyme active sites; (2) they contain a higher metalcoordination number compared with naturally occurring enzymes; and(3) they do not have access to second coordination shell residuesthat provide substantial rate enhancements in enzymes. Additionally,the critical structural and mechanistic information required for thedevelopment of the next generation of synthetic metallopeptidasescannot be readily obtained through existing experimental techniques.This is because most experimental techniques cannot follow the individualchemical steps in the catalytic cycle due to the fast rate of enzymes.They are also limited by the fact that the diamagnetic d10Zn(II) center is silent to electronic, electron spin resonance,and 67Zn NMR spectroscopies. [ABSTRACT FROM AUTHOR]

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (HO and -OH) have been investigated. In addition, the effects of the substrate ( l-leucine- p-nitroanilide → l-leucyl- p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1-Zn2 and Mg1-Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with l-leucine- p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for l-leucyl- p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0-19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1-Zn2, Mg1-Zn2, and Mg1-Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases. [ABSTRACT FROM AUTHOR]