Journal articles: 'S2 subsite'

1

CEZARI, Maria Helena S., Luciano PUZER, Maria Aparecida JULIANO, Adriana K. CARMONA, and Luiz JULIANO. "Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides." Biochemical Journal 368, no. 1 (November 15, 2002): 365–69. http://dx.doi.org/10.1042/bj20020840.

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We have examined in detail the specificity of the subsites S1, S2, S1′ and S2′ for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Dnp (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or ∊-amino-Dnp-Lys, as the fluorescence donor—receptor pair. The higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. The subsite S1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P1 had lower Km values. Despite the presence of Glu245 at S2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P2 was hydrolysed better than that containing an arginine residue. S1′ is essentially a hydrophobic subsite, and S2′ has particular preference for phenylalanine or tryptophan residues.

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2

Dion, Natalie, Paul Cohen, Philippe Crine, and Guy Boileau. "Characterisation of neprilysin (EC 3.4.24.11) S2 ′ subsite." FEBS Letters 411, no. 1 (July 7, 1997): 140–44. http://dx.doi.org/10.1016/s0014-5793(97)00681-9.

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Meeprasert, Arthitaya, Supot Hannongbua, Nawee Kungwan, and Thanyada Rungrotmongkol. "Effect of D168V mutation in NS3/4A HCV protease on susceptibilities of faldaprevir and danoprevir." Molecular BioSystems 12, no. 12 (2016): 3666–73. http://dx.doi.org/10.1039/c6mb00610h.

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LECAILLE, Fabien, Enrico WEIDAUER, Maria A. JULIANO, Dieter BRÖMME, and Gilles LALMANACH. "Probing cathepsin K activity with a selective substrate spanning its active site." Biochemical Journal 375, no. 2 (October 15, 2003): 307–12. http://dx.doi.org/10.1042/bj20030468.

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The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu67 which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2′ binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN2ph [where Abz represents o-aminobenzoic acid and EDN2ph (=EDDnp) represents N-(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly–Gly bond by cathepsin K (kcat/Km=426000 M−1·s−1). On the other hand, Abz-HPGGPQ-EDN2ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr67→Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN2ph to cathepsin K primarily depends on the S2 and S2′ subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN2ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN2ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.

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Kenjale, Roma, Guoyu Meng, Doran L. Fink, Twyla Juehne, Tomoo Ohashi, Harold P. Erickson, Gabriel Waksman, and Joseph W. St. Geme. "Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter." Infection and Immunity 77, no. 11 (August 17, 2009): 4704–13. http://dx.doi.org/10.1128/iai.00598-09.

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ABSTRACT Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapβ C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.

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Couvineau, Pierre, Hugo De Almeida, Vincent Leroux, Bernard Roques, Bernard Maigret, Catherine Llorens-Cortes, and Xavier Iturrioz. "Structural insight into the catalytic mechanism and inhibitor binding of aminopeptidase A." Biochemical Journal 477, no. 21 (November 5, 2020): 4133–48. http://dx.doi.org/10.1042/bcj20200307.

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Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2′ subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2′ subsite of human APA established various types of interactions in major part with the P2′ residue but also with the P1′ residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

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Mishra, Prasunkumar J., Chandran Ragunath, and Narayanan Ramasubbu. "The Mechanism of Salivary Amylase Hydrolysis: Role of Residues at Subsite S2′." Biochemical and Biophysical Research Communications 292, no. 2 (March 2002): 468–73. http://dx.doi.org/10.1006/bbrc.2002.6682.

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8

Brömme, D., and K. Okamoto. "The Baculovirus Cysteine Protease Has a Cathepsin B-like S2-Subsite Specificity." Biological Chemistry Hoppe-Seyler 376, no. 10 (January 1995): 611–16. http://dx.doi.org/10.1515/bchm3.1995.376.10.611.

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Sturrock, Edward D., Lizelle Lubbe, Gyles E. Cozier, Sylva L. U. Schwager, Afolake T. Arowolo, Lauren B. Arendse, Emma Belcher, and K. Ravi Acharya. "Structural basis for the C-domain-selective angiotensin-converting enzyme inhibition by bradykinin-potentiating peptide b (BPPb)." Biochemical Journal 476, no. 10 (May 31, 2019): 1553–70. http://dx.doi.org/10.1042/bcj20190290.

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Abstract Angiotensin-converting enzyme (ACE) is a zinc metalloprotease best known for its role in blood pressure regulation. ACE consists of two homologous catalytic domains, the N- and C-domain, that display distinct but overlapping catalytic functions in vivo owing to subtle differences in substrate specificity. While current generation ACE inhibitors target both ACE domains, domain-selective ACE inhibitors may be clinically advantageous, either reducing side effects or having utility in new indications. Here, we used site-directed mutagenesis, an ACE chimera and X-ray crystallography to unveil the molecular basis for C-domain-selective ACE inhibition by the bradykinin-potentiating peptide b (BPPb), naturally present in Brazilian pit viper venom. We present the BPPb N-domain structure in comparison with the previously reported BPPb C-domain structure and highlight key differences in peptide interactions with the S4 to S9 subsites. This suggests the involvement of these subsites in conferring C-domain-selective BPPb binding, in agreement with the mutagenesis results where unique residues governing differences in active site exposure, lid structure and dynamics between the two domains were the major drivers for C-domain-selective BPPb binding. Mere disruption of BPPb interactions with unique S2 and S4 subsite residues, which synergistically assist in BPPb binding, was insufficient to abolish C-domain selectivity. The combination of unique S9–S4 and S2′ subsite C-domain residues was required for the favourable entry, orientation and thus, selective binding of the peptide. This emphasizes the need to consider factors other than direct protein–inhibitor interactions to guide the design of domain-selective ACE inhibitors, especially in the case of larger peptides.

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Farley, P. C., M. G. Shepherd, and P. A. Sullivan. "The purification and properties of yeast proteinase B from Candida albicans." Biochemical Journal 236, no. 1 (May 15, 1986): 177–84. http://dx.doi.org/10.1042/bj2360177.

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A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.

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Abramić, Marija, Zrinka Karačić, Maja Šemanjski, Bojana Vukelić, and Nina Jajčanin-Jozić. "Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III." Biological Chemistry 396, no. 4 (April 1, 2015): 359–66. http://dx.doi.org/10.1515/hsz-2014-0247.

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Abstract Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.

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12

Szabó, András, Moh’d A. Salameh, Maren Ludwig, Evette S. Radisky, and Miklós Sahin-Tóth. "Tyrosine Sulfation of Human Trypsin Steers S2’ Subsite Selectivity towards Basic Amino Acids." PLoS ONE 9, no. 7 (July 10, 2014): e102063. http://dx.doi.org/10.1371/journal.pone.0102063.

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13

Kubo, M., K. Itoh, K. Nishikawa, F. Hasumi, and K. Inouye. "Mechanism of thermostability in thermolysin – analysis of subsite S2 mutant enzymes of thermolysin." Letters in Applied Microbiology 28, no. 6 (June 1999): 431–34. http://dx.doi.org/10.1046/j.1365-2672.1999.00553.x.

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Harada, Minoru, Katsuhiko Fukasawa, B. Yukihiro Hiraoka, Makio Mogi, Alfred Barth, and Klaus Neubert. "Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 830, no. 3 (August 1985): 341–44. http://dx.doi.org/10.1016/0167-4838(85)90293-6.

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Brömme, D., J. L. Klaus, K. Okamoto, D. Rasnick, and J. T. Palmer. "Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L." Biochemical Journal 315, no. 1 (April 1, 1996): 85–89. http://dx.doi.org/10.1042/bj3150085.

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Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest kinact/Ki values exceed 107 M-1·s-1 for cathepsin S and 105 M-1·s-1 for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P2 residue was synthesized. Leucine in the P2 position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L. Cathepsins O2 and S share a decreased accessibility towards P2 hydrophobic non-branched residues such as aminohexanoic acid (norleucine), methionine and oxidized methionine, but are distinguished by their different affinity towards phenylalanine in the P2 position. In contrast, cathepsin S accepts a broader range of hydrophobic residues in its S2 subsite than cathepsins O2 and L. The primary specificity-determining subsite pocket S2 in cathepsin O2 appears to be spatially more restricted than those of cathepsins S and L.

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ALVES, Marcio F. M., Luciano PUZER, Simone S. COTRIN, Maria Aparecida JULIANO, Luiz JULIANO, Dieter BRÖMME, and Adriana K. CARMONA. "S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates." Biochemical Journal 373, no. 3 (August 1, 2003): 981–86. http://dx.doi.org/10.1042/bj20030438.

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We have systematically examined the S3 to S3′ subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3–S1 subsite requirements are more restricted than those of S1′–S3′. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg–Gly and Gly–Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.

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Gour-Salin, B. J., P. Lachance, M. C. Magny, C. Plouffe, R. Ménard, and A. C. Storer. "E64 [trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of S2 subsite interactions." Biochemical Journal 299, no. 2 (April 15, 1994): 389–92. http://dx.doi.org/10.1042/bj2990389.

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A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) and benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) were synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if selectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter kinac./Ki and that the specificity of these analogues does not parallel that observed for substrates. This is particularly true in the case of the benzyl ester derivatives where the deviation from substrate-like behaviour is more important than with the benzyl amide derivatives. The results suggest that the amide proton of the benzyl amide group in HO-Eps-AA-NHBzl interacts in the S2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors. The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S2 subsite of the enzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the order of inactivation rates with chloromethane inhibitors (which are believed to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin substrates. In conclusion, the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E64 analogues in a rational manner.

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Kurbanov, Elbek K., Ting-Lan Chiu, Jonathan Solberg, Subhashree Francis, Kimberly M. Maize, Jenna Fernandez, Rodney L. Johnson, et al. "Probing the S2′ Subsite of the Anthrax Toxin Lethal Factor Using Novel N-Alkylated Hydroxamates." Journal of Medicinal Chemistry 58, no. 21 (October 29, 2015): 8723–33. http://dx.doi.org/10.1021/acs.jmedchem.5b01446.

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Cho, Sang Woo, Nam-june Kim, Myung-Un Choi, and Whanchul Shin. "Structure of aspergillopepsin I fromAspergillus phoenicis: variations of the S1′–S2 subsite in aspartic proteinases." Acta Crystallographica Section D Biological Crystallography 57, no. 7 (June 21, 2001): 948–56. http://dx.doi.org/10.1107/s0907444901005972.

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20

Turk, Dŭsan, Boris Turk, and Vito Turk. "Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?" Biochemical Society Symposia 70 (September 1, 2003): 15–30. http://dx.doi.org/10.1042/bss0700015.

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Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.

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Yates, Christopher J., Geoffrey Masuyer, Sylva L. U. Schwager, Mohd Akif, Edward D. Sturrock, and K. Ravi Acharya. "Molecular and Thermodynamic Mechanisms of the Chloride-dependent Human Angiotensin-I-converting Enzyme (ACE)." Journal of Biological Chemistry 289, no. 3 (December 2, 2013): 1798–814. http://dx.doi.org/10.1074/jbc.m113.512335.

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Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

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CHAN, Victor J., Paul M. SELZER, James H. McKERROW, and Judy A. SAKANARI. "Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease." Biochemical Journal 340, no. 1 (May 10, 1999): 113–17. http://dx.doi.org/10.1042/bj3400113.

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The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

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Lecaille, Fabien, Youngchool Choe, Wolfgang Brandt, Zhenqiang Li, Charles S. Craik, and Dieter Brömme. "Selective Inhibition of the Collagenolytic Activity of Human Cathepsin K by Altering Its S2 Subsite Specificity†." Biochemistry 41, no. 26 (July 2002): 8447–54. http://dx.doi.org/10.1021/bi025638x.

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CHAN, Victor J., Paul M. SELZER, James H. MCKERROW, and Judy A. SAKANARI. "Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease." Biochemical Journal 340, no. 1 (May 15, 1999): 113. http://dx.doi.org/10.1042/0264-6021:3400113.

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Kowlessur, D., C. M. Topham, E. W. Thomas, M. O'Driscoll, W. Templeton, and K. Brocklehurst. "Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain." Biochemical Journal 258, no. 3 (March 15, 1989): 755–64. http://dx.doi.org/10.1042/bj2580755.

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1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2′-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2′-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

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Patel, M., I. S. Kayani, W. Templeton, G. W. Mellor, E. W. Thomas, and K. Brocklehurst. "Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain." Biochemical Journal 287, no. 3 (November 1, 1992): 881–89. http://dx.doi.org/10.1042/bj2870881.

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1. 2-(N′-Acetyl-D-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound IV) (m.p. 59 degrees C; [alpha]D20 -6.6 degrees (c 1.2 in methanol)) was synthesized. 2. The results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group (Cys-25) of papain (EC 3.4.22.2) together with analogous kinetic data for the reactions of related time-dependent inhibitors, notably the L-enantiomer of compound (IV) (compound III) and the L- and D-enantiomers of 2-(N′-acetylphenylalanylamino)ethyl 2′-pyridyl disulphide (compounds I and II respectively), were used to assess the contributions of the (P1)-NH ... O = C < (Asp-158) and (P2) > C = O ... H-N-(Gly-66) hydrogen bonds and enantiomeric P2-S2 hydrophobic contacts in two manifestations of dynamic molecular recognition in papain-ligand association: (a) signalling to the catalytic-site region to provide for a (His-159)-IM(+)-H-assisted transition state and (b) the dependence of P2-S2 stereoselectivity on hydrogen-bonding interactions outside the S2 subsite. The analysis involved determination of the reactivities of individual ionization states of the reactions (pH-independent rate constants, k) and associated macroscopic pKa values and difference kinetic specificity energies (delta delta GKS = -RT1n(k1/k2), where k1 is the pH-independent second-order rate constant for reaction with one inhibitor and k2 is the analogous rate constant in the same ionization state for reaction with another inhibitor so that, when the structural change provides that k2 > k1, delta delta GKS is positive. 3. The kinetic data further illuminate the nature of the interdependence of binding interactions in papain first noted by Kowlessur, Topham, Thomas, O'Driscoll, Templeton & Brocklehurst [(1989) Biochem. J. 258, 755-764] in the S2 subsite, S1-S2 intersubsite and catalytic-site regions. Of particular note is the apparent dependence of the binding of the N-Ac-D-Phe moiety on the binding of the leaving group to (His-159)-Im+H and the fact that the resulting rate enhancement is more effective when (P1)-N-H is absent than when it is present. This result revealed by kinetic analysis goes beyond the conclusion suggested by model building that it is possible to make all of the binding contacts in complexes involving the D-enantiomers [(II) and (IV)] as in those involving the L-enantiomers [(I) and (III)].(ABSTRACT TRUNCATED AT 400 WORDS)

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27

HEMERLY, Jefferson P., Vitor OLIVEIRA, Elaine DEL NERY, Rory E. MORTY, Norma W. ANDREWS, Maria A. JULIANO, and Luiz JULIANO. "Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity." Biochemical Journal 373, no. 3 (August 1, 2003): 933–39. http://dx.doi.org/10.1042/bj20030342.

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We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1′, S2′ and S3′ subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg–Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg–Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1′ subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1′ resulted in substrates that were hydrolysed with Km values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high kcat and low Km values.

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28

Iakhiaev, Alexei, Wolfram Ruf, and Vijaya Mohan Rao. "The Role of Catalytic Cleft and Exosite Residues of Factor VIIa for Complex Formation with Tissue Factor Pathway Inhibitor." Thrombosis and Haemostasis 85, no. 03 (2001): 458–63. http://dx.doi.org/10.1055/s-0037-1615605.

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SummaryThe extrinsic coagulation pathway is initiated by the binding of plasma factor VIIa (VIIa) to the cell surface receptor tissue factor (TF). Formation of the TF-VIIa complex results in allosteric activation of VIIa as well as the creation of an extended macromolecular substrate binding exosite that greatly enhances proteolytic activation of substrate factor X. The catalytic function of the TF-VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI). TFPI inhibition of the TF-VIIa complex was enhanced by the presence of Xa. This study investigates the relative contribution of catalytic cleft and exosite residues in VIIa for inhibitory complex formation with TFPI. VIIa protease domain residues Q176, T239 and E296 are involved in the formation of stable inhibitor complex with free TFPI. Kinetic analysis further demonstrated a predominant role of the S2’ subsite residue Q176 for the initial complex formation with TFPI. In contrast, no significant reductions in inhibition by TFPI-Xa were found for each of the mutants in complex with phospholipid reconstituted TF. However, reduced rates of inhibition of the VIIa Gla-domain (R36) and Q176 mutant by TFPI-Xa were evident when TF was solubilized by detergent micelles. These data demonstrate docking of the TFPI-Xa complex with the macromolecular substrate exosite and the catalytic cleft, in particular the S2’ subsite. The masking of the mutational effect by the presence of phospholipid shows a critical importance of Xa Gla-domain interactions in stabilizing the quaternary TF-VIIa-Xa-TFPI complex.

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29

Lindahl, Peter, Daniel Ripoll, Magnus Abrahamson, John S. Mort, and Andrew C. Storer. "Evidence for the Interaction of Valine-10 in Cystatin C with the S2 Subsite of Cathepsin B." Biochemistry 33, no. 14 (April 1994): 4384–92. http://dx.doi.org/10.1021/bi00180a036.

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30

Paireder, Melanie, Ulrich Mehofer, Stefan Tholen, Andreas Porodko, Philipp Schähs, Daniel Maresch, Martin L. Biniossek, et al. "The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity." Archives of Biochemistry and Biophysics 603 (August 2016): 110–17. http://dx.doi.org/10.1016/j.abb.2016.05.017.

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31

CAMPOS, Marcelo, Constance COUTURE, Izaura Y. HIRATA, Maria A. JULIANO, Thomas P. LOISEL, Philippe CRINE, Luiz JULIANO, Guy BOILEAU, and Adriana K. CARMONA. "Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein." Biochemical Journal 373, no. 1 (July 1, 2003): 271–79. http://dx.doi.org/10.1042/bj20030287.

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The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor–acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S1´ subsite, with a strong preference for aspartate. Subsites S2´, S1 and S2 exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P2´, or valine, isoleucine or histidine in P1 precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P2 to P2´ positions, was hydrolysed by PHEX at the N-terminus of aspartate with a kcat/Km of 167 mM−1·s−1. In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N-(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.

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32

Brocklehurst, K., D. Kowlessur, G. Patel, W. Templeton, K. Quigley, E. W. Thomas, C. W. Wharton, F. Willenbrock, and R. J. Szawelski. "Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe." Biochemical Journal 250, no. 3 (March 15, 1988): 761–72. http://dx.doi.org/10.1042/bj2500761.

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1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2′-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2′-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2′-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)

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33

Brömme, D., P. R. Bonneau, P. Lachance, and A. C. Storer. "Engineering the S2 subsite specificity of human cathepsin S to a cathepsin L- and cathepsin B-like specificity." Journal of Biological Chemistry 269, no. 48 (1994): 30238–42. http://dx.doi.org/10.1016/s0021-9258(18)43803-3.

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34

Epstein, David M., and Robert H. Abeles. "Role of serine 214 and tyrosine 171, components of the S2 subsite of .alpha.-lytic protease, in catalysis." Biochemistry 31, no. 45 (November 1992): 11216–23. http://dx.doi.org/10.1021/bi00160a036.

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35

Rezaie, Alireza R. "Role of Residue 99 at the S2 Subsite of Factor Xa and Activated Protein C in Enzyme Specificity." Journal of Biological Chemistry 271, no. 39 (September 27, 1996): 23807–14. http://dx.doi.org/10.1074/jbc.271.39.23807.

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36

REID, James D., Syeed HUSSAIN, Suneal K. SREEDHARAN, Tamara S. F. BAILEY, Surapong PINITGLANG, Emrys W. THOMAS, Chandra S. VERMA, and Keith BROCKLEHURST. "Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations." Biochemical Journal 357, no. 2 (July 9, 2001): 343–52. http://dx.doi.org/10.1042/bj3570343.

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The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over < 8ps) out of papain and caricain but ‘lingers’ around the active centre of actinidin. Uniquely in actinidin, an Asp142–Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 Å) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically.

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37

Scarborough, Paula E., and Ben M. Dunn. "Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite." "Protein Engineering, Design and Selection" 7, no. 4 (1994): 495–502. http://dx.doi.org/10.1093/protein/7.4.495.

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38

Rezaie, Alireza R. "Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin." Protein Science 7, no. 2 (February 1998): 349–57. http://dx.doi.org/10.1002/pro.5560070215.

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39

Norbury, Luke J., Andrew Hung, Simone Beckham, Robert N. Pike, Terry W. Spithill, Charles S. Craik, Youngchool Choe, John V. Fecondo, and Peter M. Smooker. "Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1–P4 specificity reveals an unusual preference." Biochimie 94, no. 5 (May 2012): 1119–27. http://dx.doi.org/10.1016/j.biochi.2012.01.011.

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40

Mochalkin, Igor, and A. Tulinsky. "Structures of thrombin retro-inhibited with SEL2711 and SEL2770 as they relate to factor Xa binding." Acta Crystallographica Section D Biological Crystallography 55, no. 4 (April 1, 1999): 785–93. http://dx.doi.org/10.1107/s0907444999000359.

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Most thrombin active-site inhibitors form a short antiparallel β-strand with residues Ser214–Gly216. However, the Selectide Corp. inhibitors SEL2711 and SEL2770 bind to thrombin in a retro fashion, making a parallel β-strand with Ser214–Gly216 similar to other retro-binding inhibitors. The crystallographic structures of thrombin–hirugen complexed with SEL2711 and SEL2770, which are isostructural with the binary thrombin–hirugen complex, have been determined and refined in the 9.0–2.1 Å resolution range to final R values of 16.5 and 16.7%, respectively. The structures of the SEL2711 and SEL2770 complexes contain 131 and 104 water molecules, respectively, both of which correspond to occupancies of greater than 0.5. The L-4-amidinophenylalanyl residues of SEL2711 and SEL2770 are fixed at the S1 specificity site, utilizing favorable ionic and hydrogen-bonding interactions between the N atoms of the amidino group and the side-chain O atoms of Asp189. The Glu192 residue of thrombin adopts an extended conformation, which allows the L-cyclohexylglycyl residue in the P2 retro-binding position of the inhibitors to occupy a similar site to the P3 aspartate in thrombin platelet-receptor peptides bound to thrombin. The N-terminal acetyl group of both inhibitors is located in the S2 subsite, while the L-3-pyridyl-(3-methyl)-alanyl of SEL2711 and the L-(N,N-dimethyl)lysine of SEL2770 occupy the S3 D-Phe subsite of D-PheProArg chloromethyl ketone (PPACK) in the thrombin–PPACK complex. The two C-terminal residues of SEL2711 (leucine and proline) point into the solvent and have no electron density in the thrombin complex. Those of SEL2770 are also positioned into the solvent, but surprisingly produce weak electron density with high B values (〈B〉 = 50 Å2). Since the Selectide inhibitors are about 104 times more specific for factor Xa, modeling retro-binding to the latter suggests that the selectivity can be a consequence of interactions of the inhibitors in the S3–S4 binding subsites of factor Xa.

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41

Corvo, Ileana, Martín Cancela, Mónica Cappetta, Natalia Pi-Denis, José F. Tort, and Leda Roche. "The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen." Molecular and Biochemical Parasitology 167, no. 1 (September 2009): 41–47. http://dx.doi.org/10.1016/j.molbiopara.2009.04.005.

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42

Bagossi, Péter, Tamás Sperka, Anita Fehér, János Kádas, Gábor Zahuczky, Gabriella Miklóssy, Péter Boross, and József Tözsér. "Amino Acid Preferences for a Critical Substrate Binding Subsite of Retroviral Proteases in Type 1 Cleavage Sites." Journal of Virology 79, no. 7 (April 1, 2005): 4213–18. http://dx.doi.org/10.1128/jvi.79.7.4213-4218.2005.

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ABSTRACT The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.

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43

Ying, Q. L., A. R. Rinehart, S. R. Simon, and J. C. Cheronis. "Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes." Biochemical Journal 277, no. 2 (July 15, 1991): 521–26. http://dx.doi.org/10.1042/bj2770521.

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Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids.

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44

Lin, Ying-Chuan, Zachary Beck, Taekyu Lee, Van-Duc Le, Garrett M. Morris, Arthur J. Olson, Chi-Huey Wong, and John H. Elder. "Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease." Journal of Virology 74, no. 10 (May 15, 2000): 4710–20. http://dx.doi.org/10.1128/jvi.74.10.4710-4720.2000.

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ABSTRACT Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

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45

Villegas, Karla, Kimberly Baker-Deadmond, and Pierre F. Neuenschwander. "Mapping of Allosteric Modulator Effects in the Active Site of Factor IXa." Blood 110, no. 11 (November 16, 2007): 2695. http://dx.doi.org/10.1182/blood.v110.11.2695.2695.

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Abstract Activated factor IX (fIXa) is a vitamin K-dependent blood coagulation serine protease involved in propagation of the coagulant response through activation of fX. Maximal enzymatic and procoagulant activity of fIXa requires the presence of several cofactors; one of which is ionic calcium, which is known to bind to a site in the protease domain of fIXa as well as several sites within the light chain Gla domain region. One of the roles of calcium appears to be allosteric modulation of the fIXa active site as evidenced by an increase in enzymatic activity towards small peptidyl substrates. We and others have additionally found that certain small hygroscopic molecules can also enhance fIXa amidolytic activity. The molecular details involved in either of these effects are not well understood. Previous studies by us have shown that a pentapeptide substrate (AGRSL; the reactive site sequence of antithrombin) is hydrolyzed by fIXa in the absence of cofactors or modulators. This hydrolysis is enhanced in the presence of ionic calcium, ethylene glycol or low molecular weight heparin suggesting effects of these molecules on the immediate active site vicinity of fIXa. In order to gain insight into the potential allosteric modulation that each of these effectors may affect in fIXa, we examined the hydrolysis of four peptide libraries based on the AGRSL pentapeptide sequence, in the presence and absence of various fIXa modulators. The four libraries synthesized were XGRSL, AXRSL, AGRXL and AGRSX; where X denotes any of the possible 20 amino acids. Each of these libraries were screened for hydrolysis by fIXa under various conditions with substrates and products being identified en masse using MALDI-TOF mass spectrometry. The results suggest that ionic calcium enhances fIXa reactivity in part by modulation of the S2 subsite in fIXa. In contrast, ethylene glycol enhances fIXa activity via modulation of the S3 subsite and heparin was found to effect the overall active site region.

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46

Kim, Dong H., Zhi-Hong Li, Soo Suk Lee, Jeong-il Park, and Sang J. Chung. "A novel type of structurally simple nonpeptide inhibitors for α-chymotrypsin. Induced-fit binding of methyl 2-allyl-3-benzene-propanoate to the S2 subsite pocket." Bioorganic & Medicinal Chemistry 6, no. 2 (February 1998): 239–49. http://dx.doi.org/10.1016/s0968-0896(97)10038-4.

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47

Watermeyer, Jean M., Wendy L. Kröger, Hester G. O'Neill, B. Trevor Sewell, and Edward D. Sturrock. "Characterization of domain-selective inhibitor binding in angiotensin-converting enzyme using a novel derivative of lisinopril." Biochemical Journal 428, no. 1 (April 28, 2010): 67–74. http://dx.doi.org/10.1042/bj20100056.

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Human ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin–angiotensin–aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domain-selective inhibitor design. We have co-crystallized lisW-S, a C-domain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 Å (1 Å=0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2′ tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating C-domain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisW-S using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2′ subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.

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Porodko, Andreas, Ana Cirnski, Drazen Petrov, Teresa Raab, Melanie Paireder, Bettina Mayer, Daniel Maresch, et al. "The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities." Biological Chemistry 399, no. 10 (September 25, 2018): 1223–35. http://dx.doi.org/10.1515/hsz-2018-0186.

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Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

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Mcewan, P. A., Robert K. Andrews, and Jonas Emsley. "Glycoprotein Ibalpha Inhibitor Complex Crystal Structure at High Resolution." Blood 114, no. 22 (November 20, 2009): 774. http://dx.doi.org/10.1182/blood.v114.22.774.774.

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Abstract Abstract 774 Introduction: The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF. Methods: We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor. Conclusions: These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face. Disclosures: No relevant conflicts of interest to declare.

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Vadivel, K., Y. Kumar, G. I. Ogueli, S. M. Ponnuraj, P. Wongkongkathep, J. A. Loo, M. S. Bajaj, and S. P. Bajaj. "S2′-subsite variations between human and mouse enzymes (plasmin, factor XIa, kallikrein) elucidate inhibition differences by tissue factor pathway inhibitor -2 domain1-wild-type, Leu17Arg-mutant and aprotinin." Journal of Thrombosis and Haemostasis 14, no. 12 (November 19, 2016): 2509–23. http://dx.doi.org/10.1111/jth.13538.

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Journal articles on the topic 'S2 subsite'

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