The Structure of Papain

The data above indicate that more is involved in the reaction than just the amino acid ester serving as a nucleophile in competition with water. A specificity of the enzyme toward the amino acid ester also exists. Perhaps a better understanding of the reaction can be obtained by considering some of the properties of papain. Considerable data are available on the structure of papain, in particular its active site. His-159 is thought to be involved in catalysis as a general acid-base in which Cys-25 becomes acylated in an intermediate step (64). Schechter and Berger (27, 28) postulated from data of studies on the specificity of... 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

It can be assumed that the amino acids following this hinge region (Val 93 to Leu 447) are part of the head domain. The point of papain cleavage is at amino acid 82 27. TTie core part of the polypeptide chain is mainly folded in )3-sheets (34 %) and to a lesser extent (15 %) arranged in alpha-helical structures 7. In contrast with CBH I the core of CBH II possesses only 2 disulfide bridges (176-235 368-415) and four free sulfhydryl groups. Similarly to CBH I carboxyl functions are involved in the active center (Asp 175 and Glu 184) 28. 

Figure 17.3 Schematic representation of the design of the symmetric cathepsin K inhibitor diacylaminomethyl ketone (1,3-bis[[A/-[(phenylmethoxy)carbonyl]-L-leucyl]amino]-2-propanone), based on the crystal structures of papain bound to leupeptin (Leu-Leu-Arg-aldehyde) and to Cbz-Leu-Leu-Leu-aldehyde, and an example of its further optimization.

The relationship between the structure of the oligopeptide sequences and the rate of enzymatically catalyzed release of a drug or drug model was studied in detail. Over 50 different oligopeptide sequences were introduced into HPMA copolymers and their degradability by different enzymes studied. First, model enzymes, chymotrypsin [235, 238,239, 243,244], trypsin [245], and papain... 

The cysteinyl proteases include papain calpains I and II cathepsins , H, and L proline endopeptidase and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P, electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones, and nitriles are noteworthy since 13C-NMR spectro-... 

Crystallographic studies of native cysteine proteinases and enzyme-inhibitor complexes have been used to interpret much or the kinetic data for cysteine protemsse-caUlyzed hydrolysis of amide bonds. Analysis of the crystal structures of papain [16]. caricain [38], actinidain [56], etc. shows that these structures are closely related. The active site of all these cysteine proteinases contains the Cys-25 sulfhydryl group in close proximity to the His-159 imidazole ring nitrogens, where the latter can abstract the sulfhydryl proton to facilitate attack on the substrate amide carbonyl group [17]. 

G, Kamphuis, J. Drentii, and E. N. Baker. Thiol protease. Comparative studies based on the high-resolution structures of papain and actinidin, and an amino acid sequence information far cathenaim B and R and stem bromelain. J. MoL BioL /62 317 (19B5). 

Examination of the three-dimensional structure of papain (Lab Quip) indicated that because of the extended groove present in the vicinity of the active site residue Cys-25, it should be feasible to alkylate the sulfhydryl group with a coenzyme analog, still permitting the binding of potential substrates. 

Figure 32.3. The structure of the antibody molecule. All immunoglobulins consist of a basic subunit made up of four polypeptide chains (two light and two heavy) bound together by disulfide bonds. The variable region of the molecule contains the antigen binding site. Papain digestion yields a constant fragment (Fc) and variable fragments (Fab).

The simplest member of this family of inhibitors, the methoxy-methyl ketone (Fig. 5.8a), has been studied as a complex with papain [14]. This enzyme-inhibitor complex shows binding of the peptide portion of the inhibitor on the prime side of the active site in a manner similar to that seen for Cbz-Leu-Leu-Leu-aldehyde (Fig. 5.2d) bound to papain. In contrast to the thiohemi-acetal seen with the aldehyde, the carbonyl of the methoxy-methyl ketone (Fig. 5.8a) is quite distant from the active site thiol, with no possibility of covalent interaction. Therefore, this inhibitor meets the criteria for class I inhibition. The n-propyloxy ketone (Fig. 5.8b), which also binds on the prime side of the active site, has the ketone in close proximity to the active site thiol of cathepsin K, as seen in the structure of the inhibitor-enzyme complex [20]. Covalent attachment appears to have followed from... 

The structures of hepatitis A viral 3C proteinases complexed with tetrapeptidyl-based methyl ketone inhibitors were shown to have an episulfide cation embedded in them. The authors concluded that the mechanism of inactivation of 3G peptidases by methyl ketone inhibitors is different than those operating in serine proteinases or in papain-like cysteine peptidases <2006MI673>. 

Westrik and Wolfenden (65) have recently reported that aldehydes, with side chains similar to those comprising the acyl portion of substrates which papain effectively hydrolyzes, were very potent inhibitors of this enzyme. Umezawa and his associates (66) have also recently reported that certain microorganisms (actinomyces) produced an aldehyde, acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), which inhibits papain. The structures of some of the more effective aldehyde inhibitors of papain are shown in Figure 4. 

Properties of cathepsin L may be compared among papain-like cysteine cathepsins (36-39) for understanding its role in producing neuropeptides. Cathepsin L belongs to the CIA subfamily of Clan CA (36). Clan CA was formed based on recognition of the first cysteine protease papain. The crystal structure of papain shows two structural domains separated by an active-site cleft. The N-terminal domain is comprised of a-helices, and the C-terminal domain contains a -barrel. 

Rullman et al. (1989) studied the initial proton transfer of Cys to His with a Hartee-Fock SCF direct reaction field (DRF) method, based on the refined X-ray structure of papain (Kamphuis et al., 1984). Parts of the active site residues were represented quantum mechanically and the environment was represented by partial charges and polarizabilities. The "QM motif consisted of methanethiol (modeling Cys-25), imidazole (for His-159) and formamide (for Asn-175). All atoms at the vicinity of the active site were included, except for atoms that are too close to the active site atoms, which were deleted from the structure... 

Wang et al. (1994) analyzed by MD the roles of the "double catalytic triad" in papain catalysis, based on the structure of the enzyme, which is not completely known from crystallography (Kamphuis et al., 1984) due to the oxidation state of Cys-25 (present as cysteic acid in the crystal). Stochastic boundary MD (Brooks and Karplus, 1983) was carried out on the whole enzyme + 350 water molecules. Three "layers" were treated according to their distance from the sulfur atom of Cys-25 - atoms within 12A, atoms between 12-16A and the more distant atoms were kept fixed. CHARMM forcefield was employed. The active site geometry was examined as a function of pH, for various mutual states of S-/SH and Im/ImH+. In addition, the mutations of Asp-158 (Menard et al., 1991) were studied. 

Let us first consider the structure of myosin. The results of electron microscopic studies of skeletal-muscle myosin show it to be a two-headed structure linked to a long stalk (Figure 34.2). As we saw in Chapter 33, limited proteolysis can be a powerful tool in probing the activity of large proteins. The treatment of myosin with trypsin and papain results in the formation of four fragments two SI fragments an S2 fragment, also called heavy... 

B. S. Baines and K. Brocklehurst. A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods. Biochem. J. 777 541 (1979). 

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