Papain domains

Myoglobin, hemoglobin Thermolysin domain 2 T4 phage lysozyme domain 2 Papain domain 1... 

A. Up-and-down /3 barrels Papain domain 2 Soybean trypsin inhibitor Catalase domain 1... 

Fig. 95. Papain domain 2 as an example of an up-and-down antiparallel /3 barrel, (a) a-Carbon stereo, viewed from one side of the barrel (b) backbone schematic, viewed as in a.

Fig. 66. The domains of papain, which wrap arms around each other.

CBH I 497 core-BA aa sequence in part from protein and in full from gene (cbhl), number and location of SS bridges, region of O-glycosylation, types of carbohydrate, papain cleavage site, hydrophobic cluster analysis, computer model of active site, 2D-NMR on a synthetic tail fragment, SAXS on whole CBH I, head domain and xylan/CBH I complex... 

The sequence of the domains from the N-terminus to the C-ternunus is A- The arrow indicates the site papain cleavage. 

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 4 SAXS-based model of CBH II (redrawn from ref. 31 with permission) Note that differently to CBH I the C-terminus of the chain is at the core domain and the N-terminus at the tail domain. The sequence of the domains from the N-terminus to the C-terminus is therefore A-B-B -C. B is a repeat of B with strong sequence homologies. The arrow indicates the papain cleavage site.

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. 

Figure 8. Model structure of intact CBH I as deduced from the results of small angle X-ray scattering experiments (17). The arrow indicates the proteolytic scission site (papain), dividing the core-protein (left) from the binding domain (right). A, A-domain B, B-domain N, N-terminus C, C-terminus.

Figure 12-15 Schematic drawing of the active site of a cysteine protease of the papain family with a partial structure of an acyl-enzyme intermediate in green. The thiolate-imidazolium pair of Cys 25 His 159 lies deep in the substrate-binding cleft and bridges an interface between two major structural domains, just as the Ser His pair does in serine proteases (Fig. 12-10). This may facilitate small conformational changes during the catalytic cycle. Asn 175 provides a polarizable acceptor for positive charge, helping to stabilize the preformed ion pair, and allows easy transfer of an imidazolium proton to the product of substrate cleavage. The peptide NH of Cys 25 and the side chain of Gin 19 form an oxyanion hole.

The Ca2+-dependent neutral proteases called calpains are found within the cells of higher animals. The 705-residue multidomain peptide chain of a chicken calpain contains a papain-like domain as well as a calmodulin-like domain.328 It presumably arose from fusion of the genes of these proteins. At least six calpains with similar properties are known.329 Some have a preference for myofibrillar proteins or neurofilaments.330 They presumably function in normal turnover of these proteins and may play a role in numerous calcium-activated cellular processes.331-3323... 

RNS, ribonuclease LZM, lysozyme SNS, staphylococcal nuclease LZ4, T4 lysozyme PAP, papain TLS, thermolysin, TRX, thioiedoxin FLN, flavodoxin ADH, alcohol dehydrogenase coenzyme domain AKN, adenyl kinase MDG, malate dehydrogenase TIM, triosephosphate isomerase SUB, subtilisin CPA, carboxypeptidase LDH, lactate dehydrogenase PGK, phosphoglycerate kinase GPD, glyceraldehyde 3-phosphate dehydrogenase, HKN, hexokinase. 

Papain, a protein in which the domains are very different from each other. (Reprinted by permission of Jane S. Richardson.)... 

Phylogenetic analysis, based on the primary sequences of the catalytic domains of cysteine proteases, has demonstrated a number of different clades within the trematode non-cathepsin B-like papain superfamily (Fig. 18.6 Tort et al., 1999 Park et al., 2002 Kang et al., 2004). The parasite enzymes fall into two main... 

Domain II is composed of two subdomains (Ha and lib) and represents the catalytic core of the protease. A cys at position 115 (p,-calpain) or 105 (m-calpain), a His residue at position 272 ((x-calpain) or 262 (m-calpain) and an Asn residue at position 296 ( x-calpain) or 286 (m-calpain) form the catalytic triad characteristic of cysteine proteases such as papain or cathepsins B, L, or S. Domain II, however, shares only limited sequence homology with other cysteine proteases, and is likely to have evolved from a different ancestral gene. 

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