Academic literature on the topic 'S2 subsite'

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.

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.

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.

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.

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.

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.

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.