Papain structure

Structural Formula Chymopapain Is a sulfhydryl enzyme similar to papain. Has components of molecular weight about 35,000. 

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. 

Reactive trajectories, 43-44,45, 88,90-92,215 downhill trajectories, 90,91 velocity of, 90 Relaxation processes, 122 Relaxation times, 122 Reorganization energy, 92,227 Resonance integral, 10 Resonance structures, 58,143 for amide hydrolysis, 174,175 covalent bonding arrangement for, 84 for Cys-His proton transfer in papain, 141 for general acid catalysis, 160,161 for phosphodiester hydrolysis, 191-195,... 

IgG consists of four polypeptide subunits held together by disulphide bonds. Native immunoglobulins are rather resistant to proteolytic digestion but certain enzymes have been usefiil in elucidating their structure. Papain cleaves the molecule into three fragments of similar size ... 

Because of their very complex chemical structures and heterogeneity, melanins are difficult to extract, separate, and characterize from tissues. Eumelanins are insoluble in water and organic solvents. They can be extracted from tissues with strong chemicals that are capable of removing lipids, proteins, and other tissue components but also lead to the formation of degradation products. Enzymatic procedures were developed for the isolation of eumelanins from mammalian hair and irises. The first step is sequential digestion with protease, proteinase K, and papaine in the presence... 

When the target enzyme is difficult to obtain, related enzymes could be used to provide insights in the design of novel ligands. For example, papain was used to design a class of potent cathepsin K inhibitors [33] spanning both sides of the papain active site. However, fine-tuning these inhibitors to produce more potent ones required the use of the crystal structure of cathepsin K itself [34],... 

LaLonde JM, Zhao B, Smith WW, Janson CA, DesJarlais RL, Tomaszek TA, Carr TJ, Thompson SK, Oh HJ, Yamashita DS, Veber DF, Abdel-Meguid SS. Use of papain as a model for the structure-based design of cathepsin K inhibitors crystal structures of two papain-inhibitor complexes demonstrate binding to S -subsites. J Med Chem 1998 41 4567-4576. 

Fig. 19 Structure of LA-PRX (above) and degradation of LA-PRX (below), (a) Threaded a-CDs prevent hydrolysis of PLLA in LA-PRX. (b) LA-PRX converts into LA-pPRX by peptide linkage cleavage at bulky end-capping groups through action of papain, (c) Ester bond hydrolysis in the PLLA chain begins by an exposure of PLLA to water by release of a-CDs from LA-pPRX. Reprinted from [292] with permission...

Refinement takes place by adjusting the model to find closer agreement between the calculated and observed structure factors. For proteins the refinements can yield R-factors in the range of 10-20%. An example taken from reference 10 is instructive. In a refinement of a papain crystal at 1.65-A resolution, 25,000 independent X-ray reflections were measured. Parameters to be refined were the positional parameters (x, y, and z) and one isotropic temperature factor parameter... 

Cathepsin K (Cat K) is a member of the CA1 family of lysosomal cysteine proteases. This family is comprised of 11 human members (cathepsins B, C, F, H, K, L, O, S, V, W, Z) which share a common papain-like structural fold and a conserved active site Cys-Asn-His triad of residues [1-3]. These enzymes are synthesized as pre-pro-enzymes and are converted from the catalytically inactive zymogen into the active form in acidic lysosomal environment. In some cases, cathepsins are also secreted in the active form from cells. The sequence identity of... 

Figure 4.5. Structure of myosin. Myosin comprises both light and heavy chains. The heavy chains may be cleaved by trypsin to generate light meromyosin (LMM) and heavy mero-myosin (HMM). Papain digestion of HMM yields subfragments SI and S2 each SI fragment contains an ATPase site and an actin-binding site. The light chains modify the activity of the ATPase.

During the last ten years, it has become apparent that calcium-dependent papain-like peptidases called calpains (EC 3.4.22.17) represent an important intracellular nonlysosomal enzyme system [35][36], These enzymes show limited proteolytic activity at neutral pH and are present in virtually every eukaryotic cell type. They have been found to function in specific proteolytic events that alter intracellular metabolism and structure, rather than in general turnover of intracellular proteins. Calpains are composed of two nonidentical subunits, each of which contains functional calcium-binding sites. Two types of calpains, i.e., /i-calpain and m-calpain (formerly calpain I and calpain II, respectively), have been identified that differ in their Ca2+ requirement for activation. The activity of calpains is regulated by intracellular Ca2+ levels. At elevated cytoplasmic calcium concentrations, the precursor procal-pain associates with the inner surface of the cell membrane. This interaction seems to trigger autoproteolysis of procalpain, and active calpain is released into the cytoplasm [37]. 

CBH II 447 ABB -core aa sequence in part from protein and in full from gene (cbh2), number of SS bridges, region of O-glycosylation, types of carbohydrate, papain cleavage site, hydrophobic cluster analysis, SAXS on whole CBH II and head domain, three dimensional structure of head by X-ray diffraction... 

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.

Yamamoto, A., Tomoo, K., Doi, M, Ohishi, H., Inoue, M., Ishida, T., Yamamoto, D., Tsuboi, S., Okamoto, H., Okada, Y. (1992). Crystal structure of papain-succinyl-Gbi-Val-Val-Ala-Ala-p-nitroaniUde complex at 1.7-A resolution noncovalent binding mode of a common sequence of endogenous thiol protease inhibitors. Biochemistry 31,11305-11309. 

In an attempt to separate the domains from the cores, we used limited degradation with several proteases. CBH I (65 kda) and CBH II (58 kda) under native conditions could only be cleaved successfully with papain (15). The cores (56 and 45 kda) and terminal peptides (11 and 13 kda) were isolated by affinity chromatography (15,16) and the scission points were determined unequivocally. The effect on the activity of these enzymes was quite remarkable (Fig. 7). The cores remained perfectly active towards soluble substrates such as those described above. They exhibited, however, a considerably decreased activity towards native (microcrystalline) cellulose. These effects could be attributed to the loss of the terminal peptides, which were recognized as binding domains, whose role is to raise the relative concentration of the intact enzymes on the cellulose surface. This aspect is discussed further below. The tertiary structures of the intact CBH I and its core in solution were examined by small angle X-ray scattering (SAXS) analysis (17,18). The molecular parameters derived for the core (Rj = 2.09 mm, Dmax = 6.5 nm) and for the intact CBH I (R = 4.27 nm, Dmax = 18 nm) indicated very different shapes for both enzymes. Models constructed on the basis of these SAXS measurements showed a tadpole structure for the intact enzyme and an isotropic ellipsoid for the core (Fig. 8). The extended, flexible tail part of the tadpole should thus be identified with the C-terminal peptide of CBH I. 

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