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Papain-Like Proteases: Applications of Their Inhibitors

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Papain-Like Proteases: Applications of Their Inhibitors African Journal of Biotechnology Vol. 6 (9), pp. 1077-1086, 2 May 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals Review Papain-like proteases: Applications of their inhibitors Vikash K. Dubey1*, Monu Pande2, Bishal Kumar Singh1 and Medicherla V. Jagannadham2 1Department of Biotechnology, Indian Institute of Technology, Guwahati- 781039, Assam, India. 2Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. Accepted 5 February, 2007 Proteases are one of the most important classes of enzyme and expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. The protease family has drawn special attention for drug target for cure of several diseases such as osteoporosis, arthritis and cancer. Many proteases from various sources are being studied extensively with respect to activity, inhibition and structure. In this review, we hope to bring together the information available about the proteases with particular emphasis on papain-like plant cysteine proteases. Besides, protease inhibitors and their potential utilities are also discussed. Key words: Proteases, plant latex, reaction mechanism and protease inhibitors. INTRODUCTION Proteolytic enzymes are of widespread interest because shows evidence of their evolutionary relationship by their of there industrial application and because they have similar tertiary structures, by the order of catalytic resid- been implicated in the design and synthesis of thera- ues in their sequences, or by common sequence motifs peutic agents (Neurath, 1989). With the advent of mole- around the catalytic residues. The proteases have been cular biology, proteolytic enzymes have become a fertile organized in to evolutionary families and clans by and exciting field of basic as well as applied research. Rawlings and Barrett (1993, 1994), which led to develop- Identification of novel genes encoding proteases has ment of MEROPS database of proteases. MEROPS considerably increased our knowledge of proteases and database (http://merops.sanger.ac.uk) includes listing of provided fresh imputes. The proteases have different cel- all peptidase sequences from different families and clans. lular distribution and intracellular localization which may Each new updates adds new members and families. contribute to defining specific functional roles for some of Some representative family and clans of cysteine, serine these proteases. and threonine proteases are listed in Table 1. The related Proteases have been divided into six mechanistic families are grouped into clans, which contains all the classes by the International Union of Biochemistry. These peptidase that arose from a single evolutionary origin. include the cysteine, serine, aspartic, metalloprotease, The designation of family follows the catalytic type, serine threonine and unknown type (Enzyme nomenclature, (S), cysteine (C), or threonine (T). However, some of the 1992). The threonine protease is the most recently disco- clans are mixed type and contains families with two vered (Seemuller et al., 1995). Each class has a charac- catalytic types or more catalytic types and designated teristic set of functional amino acid residues arranged in a with the letter “P”. The cysteine protease family compri- particular configuration to form the active site. The diffe- ses six major families: the papain family, calpains, clostri- rent proteases class includes distinct families and the pains, streptococcal cysteine proteases, viral cysteine members from different family differ from each other in proteases and most recently established, caspases (also amino acid sequence despite a common active site geo- called apopains). Overall, twenty families of cysteine metry and enzymatic mechanism. Family of peptidases peptidases have been recognized (Rawlings and Barrett, 1994). The order of cysteine and histidine residues (Cys/His or His/Cys) in the linear sequence differs between families. The families C1, C2 and C10 can be *Corresponding author. E-mail: [email protected]. Tel: +91- described as papain-like, C3, C4, C5, C6, C7, C8, C9, 361-2582203. Fax: +91-361-2582249. C16, C18 and C21 are represented in viruses while C11, 1078 Afr. J. Biotechnol. C15 and C20 are from bacterial source. and viral cysteine proteases with the trypsin-fold are classified as β-proteins. The proteasome subunits are α + β proteins composed mainly of antiparallel β-sheets with CATALYTIC MECHANISM segregated α and β regions. The group of cysteine proteases with papain, cruzain, and cathepsin also has Hydrolysis of a peptide bond is an energetically favorable this structure. The subtilisins and caspases are members reaction, but extremely slow (Wolfenden and Snider, of the α/β group of proteins with parallel βs heets (β-α-β 2001). The active site residues of serine, cysteine, and units). All known cysteine proteases can be grouped in at threonine proteases are shown in Figure 1A. The active least 30 protein families. Each family contains proteins site residues in all class of proteases have many with similar amino acid sequences and evolutionarily mechanistic features in common. Each enzyme has an conserved sequence motifs, which reflects the family active site nucleophile and a basic residue, which can members' similar 3D structures. also function as a general acid in the catalytic mecha- Three-dimensional structure has been elucidated for nism. The transition states for serine, cysteine, and threo- papain, a representative member of papain-like cysteine nine proteases all involve formation of a tetrahedral proteases (Drenth et al., 1971; Kamphuis et al., 1984), intermediate shown in Figure 1B. The oxyanion of the as well as other members like actinidin (Baker, 1980), tetrahedral intermediate is frequently stabilized by inter- calotropin (Heinemann et al., 1982), cathepsin B (Musil et action with several hydrogen bond donors, which is al., 1991), caricain (Pickersgill et al., 1991), glycyl commonly referred to as the oxyanion hole. The oxyanion endopeptidase or papaya proteinase IV (O’Hara et al., hole of serine proteases is usually quite rigid and involves 1995), chymopapain (Maes et al., 1996) and cruzain backbone peptide bond NH groups as hydrogen bond (McGrath et al., 1995) and all show bilobed molecules in donors. Interaction with the oxyanion hole is usually which catalytic site is located in a cleft between the lobes. essential for effective substrate hydrolysis. With cysteine All papain-like cysteine proteases share similar seque- proteases, the oxyanion hole does not seem to be as nces (Kamphuis et al., 1985; Kirschke et al., 1995; Berti essential and is much more flexible at least in the case of and Storer, 1995) and have similar 3-dimensional struc- the papain family. tures. The structural data provides a strong evidence that Cysteine peptidases of the papain family catalyze the all arose from a common ancestor. All known papain-like hydrolysis of peptide, amide, ester, thiol ester and thiono cysteine proteases, irrespective or origin, except cathe- ester bonds (Brocklehurst et al., 1987). The basic fea- psin C, are monomers whose structure consists of two tures of the mechanism include the formation of a cova- domains (referred to as the R- and L- domains) according lent intermediate, the acyl-enzyme, resulting from nucleo- to their right and left position in the standard view. The philic attack of the active site thiol group on the carbonyl domains fold together in the form of a closed book. The carbon of the scissile amide or ester bond of the bound interactions between the domains have hydrophobic as substrate. The first step in the reaction pathway corres- well as hydrophilic character and are specific for a ponds to the association (or noncovalent binding) of the particular enzyme. The structure of papain has been free enzyme and substrate to form the Michaelis comp- extensively studied. The two catalytic residues that is, lex. Acylation of the enzyme, with formation and release Cys 25 and His 159 in papain each from N- and C- of a first product follow this step from the enzyme, the terminal domains respectively, are present in a ‘V’ like amine R′NH2. In the following step, the acyl-enzyme shaped active site cleft situated on the top of the enzyme reacts with a water molecule to form the second product structure. Recently structure of two new of papain-like (deacylation step). Release of this product results in the cysteine proteases, ervatamin B and ervatamin C, regeneration of the free enzyme. purified in our laboratory, have been reported (Biswas et al., 2003; Thakurta et al., 2004), which also shows strong structural similarly with papain (Figure 2). STRUCTURAL FOLDS Papain has a large binding site and there are a number of interactions that exist between the enzyme and the The structural determination (x-ray of NMR) of proteases substrate over an extended region. Coupling of these is lagging considerably behind the sequence determina- substrate binding interactions to the hydrolytic process tion. MEROPS database indicates that the structures of occurring at the active site is an important aspect of majority of proteases are not yet available and opens a catalysis. Schechter and Berger (1967) proposed that the wide area of studies. However, the available structures active site of papain contained seven subsites each show high degree of variability. The proteases seem to capable of accommodating a single amino acid residue of be distributed into all
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