Cysteine hydrolases mechanism

The previous chapter offered a broad overview of peptidases and esterases in terms of their classification, localization, and some physiological roles. Mention was made of the classification of hydrolases based on a characteristic functionality in their catalytic site, namely serine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallopeptidases. What was left for the present chapter, however, is a detailed presentation of their catalytic site and mechanisms. As such, this chapter serves as a logical link between the preceding overview and the following chapters, whose focus is on metabolic reactions. 

These three catalytic functionalities are similar in practically all hydrolytic enzymes, but the actual functional groups performing the reactions differ among hydrolases. Based on the structures of their catalytic sites, hydrolases can be divided into five classes, namely serine hydrolases, threonine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallohydrolases, to which the similarly acting calcium-dependent hydrolases can be added. Hydrolases of yet unknown catalytic mechanism also exist. 

Porter, D. J. Boyd, F. L. 1991 ]. Biol. Chem. 266, 21616—21625 Mechanism of bovine liver S-adenosylhomo-cysteine hydrolase. Steady-state and pre-steadystate kinetic analysis. 

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Hydrolysis of epoxides, esters, amides, and related structures is an important biotransformation reaction that limits the therapeutic activity of many drugs and generates therapeutically active drugs from prodmg structures. In a few cases, hydrolytic reactions can generate a toxic structure. Epoxide hydrolases and esterases are members of the a/(3 hydrolase-fold family of enzymes (Morisseau and Hammock, 2005 Satoh and Hosokawa, 2006). Although their substrate specificities are radically different (e.g., lipids, peptides, epoxides, esters, amides, haloalkanes), their catalytic mechanisms are similar. All of these enzymes have an active site catalytic triad composed of a nucleophilic serine or cysteine residue (esterases/amidases), or aspartate residue (epoxide hydrolases) to activate the substrate, and histidine residue and glutamate or aspartate residues that act cooperatively in an acid—base reaction to activate a water molecule for the hydrolytic step. 

On the other hand, nitrilases operate by a completely different mechanism (Scheme 2.101). They possess neither coordinated metal atoms, nor cofactors, but act through an essential nucleophilic sulfhydryl residue of a cysteine [641, 642], which is encoded in the nitrilase-sequence motif Glu-Lys-Cys [643]. The mechanism of nitrilases is similar to general base-catalyzed nitrile hydrolysis Nucleophihc attack by the sulfhydryl residue on the nitrile carbon atom forms an enzyme-bound thioimidate intermediate, which is hydrated to give a tetrahedral intermediate. After the elimination of ammonia, an acyl-enzyme intermediate is formed, which (like in serine hydrolases) is hydrolyzed to yield a carboxyhc acid [644]. 

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

Peptidases including keratinases are hydrolases able to hydrolyze peptide bonds in proteins and peptides. They are classified using three different approaches (1) the chemical mechanism of catalysis (based on the catalytic amino acid or metal ion at then-active site, represented by serine, cysteine, threonine, aspartic, asparagine, glutamic and metallocatalytic type), (2) the catalytic reaction (this type of classification depends on the selectivity for the bonds that the peptidases will hydrolyze), and (3) the molecular structure and homology. In this latter approach, amino acid... 

A. Lodola, D. Branduardi, M. De Vivo, L. Capoferri, M. Mor, D. Piomelli, and A. Cavalli, PLoS One, 7(2), c32397 (2012). A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations. 

Classification of this enzyme as a serine, cysteine, aspartic, or metallo-dependent enzyme [50] is somewhat problematic because occasionally inhibitors from the same class led to contradictory results. The observed effects suggest, however, that both metal ions and sulfhydryl groups may play a major role in the hydrolytic mechanism. Therefore, most probably the enzyme is a metallocysteine hydrolase. 

---