Recognition of Active and Inactive Catalytic Triads: a Template Based Approach

International Journal of Biological Macromolecules 46 (2010) 317–323

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International Journal of Biological Macromolecules

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Recognition of active and inactive catalytic triads: A template based approach

Vikas Gupta a,1, N.A. Udaya Prakash a,1, V. Lakshmi b, R. Boopathy b, J. Jeyakanthan c, D. Velmurugan d, K. Sekar a,∗ a Bioinformatics Centre, (Centre of Excellence in Structural Biology and Bio-computing), Indian Institute of Science, Bangalore 560 012, India b School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India c National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu Science Park, Hsinchu 30076, Taiwan d Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India article info abstract

Article history: It is well established that a sequence template along with the database is a powerful tool for identifying the Received 30 November 2009 biological function of proteins. Here, we describe a method for predicting the catalytic nature of certain Received in revised form 13 January 2010 proteins among the several protein structures deposited in the Protein Data Bank (PDB). For the present Accepted 14 January 2010 study, we considered a catalytic triad template (Ser-His-Asp) found in serine proteases. We found that a Available online 25 January 2010 geometrically optimized active site template can be used as a highly selective tool for differentiating an active protein among several inactive proteins, based on their Ser-His-Asp interactions. For any protein Keywords: to be proteolytic in nature, the bond angle between Ser O␥–Ser H␥···His N␧2 in the catalytic triad needs Catalytic triad ◦ ◦ ␥··· ␧2 Serine protease to be between 115 and 140 . The hydrogen bond distance between Ser H His N is more ﬂexible in ␦1··· ␦1 Hydrogen bond network nature and it varies from 2.0 Å to 2.7 Å while in the case of His H Asp O , it is from 1.6 Å to 2.0 Å. 2 Three-dimensional structure In terms of solvent accessibility, most of the active proteins lie in the range of 10–16 Å , which enables Solvent accessibility easy accessibility to the substrate. These observations hold good for most catalytic triads and they can Intramolecular distances be employed to predict proteolytic nature of these catalytic triads. © 2010 Elsevier B.V. All rights reserved.

1. Introduction form catalytic cleavage of an appropriate bond of the substrate. Such a triad involvement was first identified in chymotrypsin and A protein’s sequence motifs and templates are important tools subtilisin [5,6], results in cleaving the peptide bond. Proteolytic for the identification or prediction of its biological function and enzymes are widely distributed in nature and mediate a wide range tertiary structure [1–3]. Normally, the templates are derived from of physiological responses from the onset of blood clotting [7] to the one-dimensional protein sequence signature by analyzing and the digestion [8] of proteins in the alimentary system. They also comparing the information from the known protein structures, play a major role in the tissue destruction associated with arthri- especially the data generated from sequence alignments and pat- tis, pancreatitis, and pulmonary emphysema. Such enzymes are tern matching techniques (for example [4]). Thus, the derived highly specific for its own substrates and the specificity is in terms three-dimensional template will provide a quantitative descrip- of identifying the residue of the substrate that fits into the bind- tion about the relative dispositions of the key residues in the active ing pocket of the enzyme, present immediately adjacent to scissile site of the enzyme based on their three-dimensional atomic coor- amide bond. A detailed explanation on the role of catalytic triad dinates. Such a template, when generated can be used to scan the has been published elsewhere, which is a comprehensive review databases of known protein structures to identify the catalytic cen- of the structural basis of substrate specificity in serine proteases ters. [9]. Another group has performed an extensive steric comparison The three residues of a catalytic triad (Ser-His-Asp of serine pro- of the catalytic site residues in serine proteases [10]. They analyzed tease), occur far apart in the primary structure of the enzyme, come the differences in the relative conformations of the Ser-His-Asp together in a specific conformation called the active site and per- residues by performing root mean square (rms) fits for all occur- rences. On the basis of the differences and similarities, they were able to classify the serine proteases into chymotrypsin and sub- stilisin families. They also found several examples of Ser-His-Asp ∗ Corresponding author at: Bioinformatics Centre, (Centre of Excellence in Struc- triads in non-proteolytic proteins in similar conformations to that tural Biology and Bio-computing), 101, Raman Building, Indian Institute of Science, of serine proteases [11]. Bangalore 560 012, Karnataka, India. Tel.: +91 80 22933059/22932469/23601409; Artymiuk et al. [12] have used graph-theory approach for fax: +91 80 23600683/23600551. E-mail address: [email protected] (K. Sekar). the identification of catalytic triad residues based on the three- 1 These authors equally contributed to this work. dimensional patterns of amino acid side chains in protein

0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.01.011 318 V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323 structures. A different structural comparison of the serine proteases using a less specific technique has been performed by Fischer et al. [13]. Their method derived from geometric hashing is widely used in the field of computer vision research. In this method, all C␣ atoms in the backbone are considered as points in space and protein structures are compared purely on the geometric relationships of these points. They were able to identify structural similarities between the active site of the trypsin-like and the subtilisin-like serine proteases based solely on the similarities of C␣ geometries of their constituent catalytic residues. Further, Cai et al. have employed support vector machines to identify catalytic triads present in serine hydrolase family [14]. A method called Comparative Molecular Field Analysis (CoMFA) which provides statistical and graphical models that relate the properties of molecules (including biological activity) to their structures. These models are then used to predict the properties or activity of the novel compounds [15]. Apart from the serine proteases, the Ser-His-Asp catalytic triad also occurs in several lipases, which are responsible for hydrolyzing triglycerides into diglycerides and subsequently monoglycerides and free fatty acids. The catalytic mechanism in such lipases is also mediated by the catalytic serine of the triad [8,16]. The catalytic triad is buried beneath a short stretch of helix, known as the “lid”. A number of crystallographic studies have confirmed the hypothe- sis that the lid is displaced during activation of lipases [16,17], being rolled back as a rigid body into the hydrophilic trench filled previ- ously by water molecules, thereby exposing the active site. Thus, the present study is carried out to better understand the relative conformational orientation of the catalytic triad residues. Our analysis differs substantially from the earlier studies [10,12–15].We aim to throw light on the geometric and physiochemical features of the catalytic triad of serine proteases and employ the results to differentiate between active and inactive catalytic triads. Thus, we derived templates of catalytic triads that are present in proteases. Further, we analyzed all protein structures in which the Ser O␥ atom and the Asp O␦1 and O␦2 atoms are present at a distance of 3.6 Å or less with the N␦1 and N␧2 atoms of the histidine residue. The hydrogen bond networks and the orientation of serine, aspartate and histidine residues of the catalytic triad were also analyzed. Further, we have demonstrated that once a three-dimensional template is derived from the PDB file, it can be employed to provide valuable information for predicting the catalytic nature of a protein.

2. Materials and methods

A total of 58,083 three-dimensional structures (as of June, 2009) were deposited in the PDB [18]. About 4655 structures in the PDB contain interactions between the three (Ser-His-Asp) amino acid residues. Among the residues present in the catalytic triad, a cut-off distance of 3.6 Å was considered between the Ser O␥ atom and Asp Fig. 1. (a) The ball and stick representation of the active catalytic triad identified O␦1 or O␦2 atom with His N␦1 or N␧2 atom. In about 2878 structures, in plasminogen activator protein (PDB-id: 1C5S). Interactions between Ser O –Ser H ···His Nε2 and His Nı1–His Hı1Asp···Oı2 atoms are shown. (b) The ball and stick the Ser O␥ atom was found to be hydrogen bonded with N␧2 of the representation of the active catalytic triad identified in Phospholipase A2 (PDB-id: histidine residue (Fig. 1a) and 1777 structures were found to have 1SQZ). The interactions between Ser O␥–Ser H␥–His N␦1 and HisN␧2–His H␧2···Asp ␥ ␦ interaction between Ser O and N 1 of the histidine residue (Fig. 1b). O␦2 atoms are shown. The histidine residue has an imidazole ring in which the hydrogen atom can shuttle from N␦1 to N␧2 and can be present in any of the two nitrogen atoms. Out of the total number of structures (4655) in further divided into two groups (group 1 proteins contain a cat- the dataset, 1131 were considered as false positives since they con- alytic triad and participate in proteolysis and group 2 proteins tain the Ser-His-Asp motif and have no associated catalytic activity. contain a catalytic triad but do not participate in proteolysis). The In the case of redundant structure, only the higher resolution (reso- proteins were divided into two groups using SCOP [19], CATH lution better than or equal to 3.0 Å) structures were considered for [20] and The Gene Ontology database [21]. The SCOP and CATH the analysis. The interatomic distances and bond angles were cal- databases were used to classify proteins into different classes, culated on the basis of the three-dimensional atomic coordinates based on their respective families and sub-families, while the Gene available in the PDB files. Ontology database was used to ascertain their molecular func- The three-dimensional structures that fit with the above men- tion. The databases SCOP and CATH provide us an idea if proteins tioned criteria were incorporated into the final dataset and were with trypsin and subtilisin-like structures perform proteolysis and V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323 319 the Gene Ontology database was used to confirm their biological activity. Thus a stringent double check was employed to increase the preciseness of our classification. Pearson correlation coefficient was calculated for the observed frequencies. The NACCESS program in MIMOX [22] was used to calculate both the residue level and the atomic level solvent accessibility of the catalytic serine residue and its O␥ atom. The GROMACS program [23] was employed for adding hydrogen in low resolution structures. The three-dimensional structures were superimposed using the freely available internet computing service 3dSS [24]. The orientations of the catalytic residues were analyzed using COOT [25] and visual- ization of the three-dimensional structures was performed using PyMOL [26].

3. Results and discussion

Several studies have elucidated that the Ser O␥ atom and the histidine residue of the catalytic triad are in juxtaposition with the substrate scissile amide bond [5,6,9]. The close association between the residues of the catalytic triad is mainly governed by their hydrogen bonds and their accessibility to water molecules. The ionization of these residues and their subsequent interactions with water molecules is again controlled by the pH of their sur- rounding medium. Moreover, for any catalytic triad to bring about catalysis of the substrate, the Ser O␥ must be a nucleophile. In order to understand the geometric and physiochemical characteristics of catalytic triads, several related studies are performed and the results are discussed in the subsequent sections.

3.1. Hydrogen bond angle

Fig. 2. Histograms showing hydrogen bond angles between Ser O␥–Ser H␥···His The strength of the hydrogen bond is influenced by several N␧2 atoms of the catalytic triad present in all proteins belonging to (a) group 1 factors like temperature, pressure, bond angle and environment (performing proteolysis) and (b) group 2 (not participating in proteolysis). (usually characterized by local dielectric constant). Further, the ideal hydrogen bond angle depends upon the orientation of the donor. Thus, an attempt has been made to estimate the degree of variation in hydrogen bond angles among the residues forming in the native state. However, the bond angle changes to 54.89◦ after the catalytic triad. Fig. 2a and b shows histograms plotted for the the addition of a ligand (Fig. 4). Similarly, in proteinase K (PDB-id: hydrogen bond angle data derived from the Ser O␥–Ser H␥···His 1P7W) the hydrogen bond angle between the Ser O␥–Ser H␥···His N␧2 atoms of the catalytic triad, present in all proteins belonging to N␧2 atoms is found to be 139.34◦ in the native state and it changes to group 1 (performing proteolysis) and group 2 (not performing pro- 74.33◦ after the addition of a ligand. Thus, it is clearly seen that the teolysis). Similarly, Fig. 3a and b shows histograms plotted for the interaction of ligands with the protein structure results in reducing hydrogen bond angle data derived from the His N␦1–His H␦1···Asp the hydrogen bond angle between Ser O␥–Ser H␥···His N␧2 atoms, O␦2 atoms of the catalytic triad, present in all proteins belonging to thereby rendering the protein inactive. group 1 and group 2. From Fig. 3a, it is observed that the hydrogen bond angle Fig. 2a and b, 3a and b provide a general picture about the varia- between the His N␦1–His H␦1···Asp O␦2 atoms should lie between tion in the hydrogen bond angle in Ser O␥–Ser H␥···His N␧2 and in 150◦ and 170◦ and from Fig. 3b, it is also observed that the hydro- His N␦1–His H␦1···Asp O␦2 atoms. Clear differences are observed on gen bond angle between the His N␦1–His H␦1···Asp O␦2 atoms of comparing Fig. 2a and b and it can be concluded that the hydrogen several inactive proteins also lie in the 150–170◦ range. Hence, the bond angle between Ser O␥–Ser H␥···His N␧2 should be between hydrogen bond angle between the His N␦1–His H␦1···Asp O␦2 atoms 115◦ and 140◦. Any protein with a catalytic triad that does not sat- cannot be used as an effective measure to differentiate between isfy this criterion is considered to be non-proteolytic in nature. The active and inactive proteins. However, as stated earlier, clear differ- difference in the hydrogen bond angles can also be attributed to ences are observed between Fig. 2a and b and hence the hydrogen the change in orientation of the serine moiety. In most of the non- bond angle between the Ser O␥–Ser H␥···His N␧2 atoms can be used proteolytic proteins, the hydrogen bond angles are either less than as a filter to differentiate between active and inactive proteins. The 115◦ or greater than 140◦ (Fig. 2b). Pearson correlation coefficients are calculated for the frequencies Few proteins in group 1, are active in their native form with plotted in Fig. 2a and b, 3a and b. The values for the active (Fig. 2a) hydrogen bond angles 75◦ and 105◦ but these proteins are suscep- and inactive (Fig. 2b) conformations of the catalytic triad is found tible to inhibition by ligands. Interaction of hydrogen atoms with to be −0.0186, which indicates a very low correlation. Further, for ligands causes the catalytic triad to lose its hydrogen bond net- frequencies plotted in Fig. 3a and b, the Pearson correlation coeffi- work and hence results in the loss of catalytic activity. Thus, the cient is found to be 0.844, which indicates a moderate correlation. loss of catalytic activity of a protein may be explained by hydrogen The results of the statistical test support the above conclusion that bond network associated disruption of interactions in the catalytic hydrogen bond angle between Ser O␥–Ser H␥···His N␧2 atoms can triad region. In trypsin (PDB-id: 1C1P), the hydrogen bond angle be employed to effectively differentiate between active and inac- between the Ser O␥–Ser H␥···His N␧2 atoms is found to be 124.32◦ tive catalytic triads. 320 V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323

Fig. 3. Histograms showing hydrogen bond angles between His N␦1–His H␦1···Asp Fig. 4. The two states of the catalytic triad identiﬁed in trypsin (PDB-id: 1C1P) are O␦2 atoms of the catalytic triad present in all proteins belonging to (a) group 1 and shown (superimposed) in ball and stick representation. The state I (cyan) and state (b) group 2. II (purple) are shown without and with the ligands, respectively. The difference in Ser H␥ positions is clearly visible.

In Fig. 5, the catalytic triad of a Trypsin structure (PDB-id: 1XVO) is shown. It is observed that the orientation of the serine side 3.2. Hydrogen bond distance chain is almost perpendicular to the imidazole ring of the histidine residue and the bond angle lies in the 120–140◦ range. The aspartate The distance between the hydrogen donor and the acceptor is residue also lies in a similar position like the serine residue, with an important factor that determines the catalytic nature of the respect to the histidine ring making a higher angle that favours active site. The hydrogen bond distances between the Ser H␥···His hydrogen bond formation. It is well known that hydrogen bond N␧2 and His H␦1···Asp O␦2 atoms are calculated. The histograms in angle criterion is one the most important feature that affects the Fig. 6a and b represent the hydrogen bond distances between the activity of proteolytic enzymes. Pauling [27] reported that enzymes Ser H␥···His N␧2 atoms present in all proteins belonging to group work by stabilizing the conformation of the transition-state com- 1 and group 2. Similarly, the histograms in Fig. 7a and b represent plex, which is achieved by hydrogen bond formation. Thus, the the distances between the His H␦1···Asp O␦2 atoms present in all present study shows the hydrogen bond angle between Ser O␥–Ser proteins belonging to group 1 and group 2. From the Fig. 6a and b, H␥···His N␧2 atoms plays a vital role in determining the proteolytic it can be observed that the hydrogen bond distance between the activity of a catalytic triad. Ser H␥···His N␧2 atoms is much more ﬂexible in nature and varies

Fig. 5. The ball and stick representation of the proper orientation that any catalytic triad must adopt to be in the active form. The catalytic triad shown is identiﬁed in trypsin (PDB-id: 1XVO). V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323 321

␥··· ␧2 Fig. 6. Histograms showing hydrogen bond distances (Å) between Ser H HisN Fig. 7. Histograms showing hydrogen bond distances (Å) between His H␦1···Asp O atoms of the catalytic triad present in all proteins belonging to (a) group 1 and (b) ␦2 atoms of the catalytic triad present all proteins belonging to (a) group 1 and (b) group 2. group 2. from 2.0 Å to 2.7 Å, while in the case of His H␦1···Asp O␦1 atoms it varies from 1.6 Å to 2.0 Å (Fig. 7a and b). between the donor and the acceptor atoms influences the catalytic The Pearson correlation coefficients are calculated for the activity. observed frequencies plotted in Fig. 6a and b, 7a and 7b.Itis The histidine to alanine mutation results in lowering the enzyme observed that for frequencies plotted for active (Fig. 6a) and inac- activity by a factor of 100,000 [29]. The position of the histidine tive (Fig. 6b) conformations, the Pearson correlation coefficient is residue in the sequence is variable, but in the three-dimensional ␧2 found to be 0.732, which indicates a low level positive correla- structure its N atom is invariably close to the serine residue. It tion. Further, for Fig. 7a and b the Pearson correlation coefficients is to be noted that the tautomeric form of the enzyme has to be ␦1 ␧2 are found to be 0.99, which indicates high level correlation. Here protonated on the N atom and not on the N atom. This obser- again, the Pearson correlation coefficients suggest that the hydro- vation is in contrast to the normal conformation observed for most gen bond distance of Ser H␥···His N␧2 atoms can differ significantly histidines, although exceptions are reported [30,31]. Thus, it can compared to that of His H␦1···Asp O␦1 atoms between active and be concluded that for proteins to be active, the hydrogen bond dis- ␥··· ␧2 ␦1··· ␦2 inactive conformations of the catalytic triads. tance between the Ser H His N and His N Asp O atoms From the above results, it can be inferred that the aspartate should be from 2.0 Å to 2.7 Å and 1.6 Å to 2.0 Å respectively. residue involved in the catalytic triad is present at the same position in all catalytic triads. Whereas, the position of the serine 3.3. Accessibility residue involved in the catalytic triad varies. A few exceptions show peaks at a distance greater than 2.7 Å (Fig. 6b). Such pro- In the present study, we found the serine residue of the catalytic teins are active in their native form but show interactions with triad to be surrounded by extra amino acid residues and hetero- ligands, which inhibit their proteolytic activity. For example, in the molecules like H2O. A serine residue along with H2O molecules is case of ␣-lytic protease (PDB-id: 2H5C) the hydrogen bond dis- present in the vicinity of the catalytic triad. Interestingly, in some tance between the Ser H␥···His N␧2 atoms is found to be 2.17 Å cases we observed that the position of the serine residue was occu- (native form), but the distance increases to 3.18 Å in the ligand pied by a threonine or a cysteine residue. These amino acid residues bound form. The above example clearly elucidates the importance are also implicated in the formation of a hydrogen bond network of hydrogen bonding distance in determining the catalytic activ- [32], which enables them to act as nucleophile during catalysis. ity of a protein. Katz et al. [28] have shown that the interaction Thus, the extra amino acid residue together with the catalytic triad between the Ser H␥ atom and an inhibitor resulted in increased dis- residues constitutes the catalytic tetrad. tance between the Ser H␥ and the His N␧2 atoms. They also observed For the serine residue to act as a nucleophile its O␥ atom should that such an interaction resulted in the loss of catalytic activity of be accessible from both the inside and the outside of the protein and the protein. Thus, it can be stated that hydrogen bonding distance this requires the absence of any intervening bulky side chains. As a 322 V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323

proteins present in groups 1 and 2 (data not shown). Whereas, the atomic level accessibility calculated for the O␥ atom of the serine provided clear differences between the proteins present in the groups 1 and 2. This proves that solvent accessibility of the catalytic Ser O␥ atom plays a vital role in determining the proteolytic nature of catalytic triads. Fig. 8a and b depicts the solvent accessibility values plotted for the Ser O␥ atom of the catalytic triad that is present in all proteins belonging to groups 1 and 2. On comparing these two ﬁgures differences in solvent accessibility, between the proteins belonging to group 1 and group 2, are clearly observed. In Fig. 8b, a large peak in the 0–3 intervals corresponds to an inactive conformation of the Ser O␥ atom. Further, a small peak observed in the same interval in Fig. 8a corresponds to protein structures that are complexed with inhibitors. The Pearson correlation coefﬁcient calculated for the observed frequencies plotted in Fig. 8a and b and is found to be 0.821, which indicates a moderate level correlation. From Fig. 8a, we also observe that most of the active proteins lie in the range of 10–6 Å2. Thus, it can be concluded that in proteins with active catalytic triads, the accessibility of active site Ser O␥ atom lies in the 10–16 Å2 range.

4. Conclusion

The proteolytic nature of proteins with the catalytic triad is governed by two major factors, namely, the orientation of the serine reside in the catalytic triad and by accessibility to the solvent. The position of the hydrogen atoms and the nature of their interactions also contribute to the activity of the protein. For any protein to be catalytic in nature, the bond angle between Ser O␥–Ser H␥···His N␧2 in the catalytic triad should lie between 115◦ and 140◦. The hydrogen bond distances between Ser H␥···His N␧2 is much more flexible in nature and it varies from 2.0 Å to 2.7 Å while in the case 2 ␥ Fig. 8. Histograms showing solvent accessibility (Å ) of Ser O atom of the catalytic of His H␦1···Asp O␦1, it is from 1.6 Å to 2.0 Å. The solvent accessi- triad present in all proteins belonging to (a) group 1 and (b) group 2. bility of the active site Ser O␥ atom lies in the range of 10–16 Å2. Further, the results obtained from the statistical tests reveal that result, glycine residues are often observed in the immediate vicinity the hydrogen bond angle between Ser O␥–Ser H␥···His N␧2 atoms of the serine residue [33–35]. In such proteins, the serine and the play a crucial role in differentiating the active and inactive catalytic glycine residues serve to form a tight hairpin turn which directs the triads. The other factors like hydrogen bond distance and accessi- serine side chain into the active site cavity. This movement serves to bility can also be employed along with the hydrogen bond angle ␥ initiate an interaction between the Ser O atom and the substrate. criterion to assure the prediction. The Ser O␥ atom of the catalytic triad is found to be mobile in nature and this phenomenon is clearly observed in wheat serine Acknowledgements carboxypeptidase II [36]. Further, the serine residue present in the catalytic triad of the Bacillus subtilis lipase is found to adopt either an The work reported is fully supported by the Department of active or an inactive conformation [37]. These two conformations Information Technology (DIT), Government of India. The authors of the serine residue arise due to the differences in the position gratefully acknowledge the facilities at the Bioinformatics Centre ␥ of its O atom. Thus, the active conformation of the serine residue and the Interactive Graphics Based Molecular Modelling (IGBMM). ␥ plays a crucial role in placing its O atom in an accessible position. Further, these two facilities are supported by the Department of The abstraction of a proton from a serine residue by a histidine Biotechnology. residue (general acid base catalysis) would be greatly favoured by a hydrogen bond between these residues [38]. Similarly, the hydro- References gen bond network between the catalytic triad residues accounts for the increased basicity of the histidine residue [39]. [1] W.R. Taylor, Protein Eng. 2 (1998) 77–86. The environment of the aspartate residue of the catalytic triad is [2] T.C. Hodgman, Comput. Appl. Biosci. 5 (1989) 1–13. [3] W.R. Taylor, D.T. Jones, Curr. Opin. Struct. Biol. 1 (1991) 327–333. highly polar and it has been proposed that the amino acid residues [4] S.S. Sheik, S.K. Aggarwal, A. Poddar, N. Balakrishnan, K. Sekar, J. Chem. Inform. present around the aspartate residue form an “aspartate hole”. Comput. Sci. 44 (2004) 1251–1256. Thus, the aspartate residue is always kept in an ionized condition [5] D.M. Blow, J.J. Birktoft, B.S. Hartley, Nature 221 (1969) 337–340. [40]. The present study identifies several proteins like those belong- [6] C.S. Wright, R.A. Alden, J. Kraut, Nature 221 (1969) 235–242. [7] K.G. Mann, Trends Biochem. Sci. 12 (1987) 229–233. ing to the Cyclophilin family that do not perform proteolysis in spite [8] D.M. Blow, Biochem. J. 110 (1968) 1–2. of possessing a catalytic triad. In all these proteins, the catalytic [9] J.J. Perona, C.S. Craik, Protein Sci. 4 (1995) 337–360. triad residues are not accessible by the substrate. Hence, accessi- [10] A. Barth, M. Wahab, W. Brandt, K. Frost, Drug Des. Discov. 10 (1993) 297–317. [11] A. Barth, K. Frost, M. Wahab, W. Brandt, H.D. Schlader, R. Frank, Drug Des. Discov. bility is considered as a major factor that influences the catalytic 12 (1994) 89–111. activity of a protein. Thus, we calculated both the residue level and [12] P.J. Artymiuk, A.R. Poirrette, H.M. Grindley, D.W. Rice, P. Willett, J. Mol. Biol. the atomic level solvent accessibility for the catalytic serine residue 243 (1994) (1994) 327–344. ␥ [13] D. Fischer, H. Wolfson, S.L. Lin, R. Nussinov, Protein Sci. 3 (1994) 769–778. and its O atom. The residue level solvent accessibility of the cat- [14] Y. Cai, G. Zhou, C. Jen, S. Lin, K. Chou, J. Theor. Biol. 228 (2004) 551–557. alytic residue serine did not show significant differences between [15] R.D. Cramer, D.E. Patterson, J.D. Bunce, J. Am. Chem. Soc. 110 (1988) 5959–5967. V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323 323

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