Cysteine hydrolases

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. 

The serine hydrolases, threonine hydrolases, and cysteine hydrolases, the attacking nucleophile of which is a serine or threonine OH group or a cysteine thiolate group, respectively, and which form an intermediate covalent complex (i. e., the acylated enzyme). Here, an activated H20 molecule enters the catalytic cycle in the second step, i.e., hydrolysis of the covalent intermediate to regenerate the enzyme. 

In addition to the stable isotope labeling ( 0 versus 0) of proteins for quantifiable proteomic analyses as described above, chemical approaches to the protein-labeUng problem have developed in great variety. These so-called affinity tags can be used to label specific side chain groups such as sulfhydryl or amino groups, active sites for serine and cysteine hydrolases and many others. This active research area has been reviewed recently by A. Leitner and W. Lindner in a Proteomics article entitled Chemistry meets proteomics The use of chemical tagging reactions for MS-based proteomics. ... 

Bound NAD+ is also present in S-adenosylhomo-cysteine hydrolase,119 120 which catalyzes the irreversible reaction of Eq. 15-14. Transient oxidation at the 3 position of the ribose ring facilitates the reaction. The reader can doubtless deduce the function that has been established for the bound NAD+ in this enzyme. However, the role of NAD in the urocanase reaction (Eq. 15-15) is puzzling. This reaction, which is the second step in the catabolism of histidine, following Eq. 14-44, appears simple. However, there is no obvious... 

Metallohydrolases are the third major class of hydrolytic enzymes. Unlike serine or cysteine hydrolases, the metallohydrolases achieve substrate hydrolysis via a zinc-activated water molecule [78-80] (Scheme 3). 

Bound NAD is also present in S-adenosylhomo-cysteine hydrolase, T20 catalyzes the irrevers-... 

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. 

Similar equations were derived for the closely related cysteine hydrolases actinidin [288, 696-698], bromelain [694, 697, 698], and ficin [697, 698]. 

Carotti, A., Raguseo, C. and Hansch, C. 1985. Quantitative structure-activity relationships of cysteine hydrolases. Ficin hydrolysis of X-phenyl-N-(methanesulfonyl) glycinates. Chem.-Biol. Interactions 52 279-288. 

L-cysteine DL- 2- amino thia2 oline- 4- c arb 0 xyUc hydrolase + racemase Pseud, thia olinophilum ... 

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

The NC-IUBMB has introduced a number of changes in the terminology following the proposals made by Barrett, Rawlings and co-workers [7] [8]. The term peptidase should now be used as a synonym for peptide hydrolase and includes all enzymes that hydrolyze peptide bonds. Previously the term peptidases was restricted to exopeptidases . The terms peptidase and protease are now synonymous. For consistency with this nomenclature, the term proteinases has been replaced by endopeptidases . To complete this note on terminology, we remind the reader that the terms cysteine endopeptidases and aspartic endopeptidases were previously called thiol proteinases and acid or carboxyl proteinases , respectively [9],... 

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. 

After an oral dose of 6 mg/kg bw to rats, approximately 38% of the dose was exhaled as CO2, 50% was excreted as metabolites in the urine and 3% was present in faeces (Gingell et al., 1985). Concentrations were highest in liver, kidney and forestomach. The initial metabolic reactions are conjugation of the epoxide with glutathione, which is probably a chemical, not enzymatic, reaction, and hydration of the epoxide by epoxide hydrolase. The major metabolites in urine are jV-acetyl-5 -(3-chloro-2-hydroxypropyl)-L-cysteine (36% of the dose) and 3-chloro-1,2-propanediol (a-chlorohydrin) (4%). 

Sakamoto, T., Tanaka, T., Ito, Y., et al. (1999) An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase evidence for novel substrate recognitionby a cysteine protease. Biochemistry 38, 11,634-11,642. 

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