Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform?

The Lachesis muta thrombin-like enzyme (LM-TL) is a single chain serine protease that shares 38% sequence identity with the serine protease domain of thrombin and also displays similar fibrinogen-clotting activity. In addition, the 228 amino acid residue LM-TL is 52% identical to trypsin, and cleaves chromogenic substrates with similar specificity. Herein we report a three-dimensional (3D) model validated experimentally for LM-TL based on these two homologous proteins of known 3D structure. Spatial modeling of LM-TL reveals a serine protease with a chymotrypsin fold presenting a hydrophobic pocket on its surface, involved in substrate recognition, and an important 90's loop, involved in restricting the LM-TL catalytic site cleft. Docking analysis showed that LM-TL would not form a stable complex with basic pancreatic trypsin inhibitor and wild-type ecotin since its 90's loop would restrict the access to the catalytic site. LM-TL formed acceptable interactions with fibrinopeptide A and a variant of ecotin; ecotin-TSRR/R in which both the primary and secondary binding sites are mutated Val81Thr, Thr83Ser, Met84Arg, Met85Arg and Asp70Arg. Furthermore, analysis of the primary structures of LM-TL and of the seven snake venom thrombin-like enzymes (SVTLEs) family reveals a subgroup formed by LM-TL, crotalase, and bilineobin, both closely related to thrombin. Therefore, LM-TL provides an initial point to compare SVTLEs with their counterparts, e.g. the mammalian serine proteases, and a basis for the localization of important residues within the little known SVTLEs family.     

Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.     

Active site mapping of trypsin,thrombin and matriptase-2 by sulfamoyl benzamidines

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands, including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate specificity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the S1 pocket of such enzymes. Six series of benzamidine derivatives (1–73) were synthesized and evaluated as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investigated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation of the hepcidin expression. Compounds 1–73 were designed to contain a fixed sulfamoyl benzamidine moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, carboxylate or second benzamidine functionality. A systematic mapping approach was performed with these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able to interact with both the S1 and S3/S4 binding sites, showed notable affinity. In branched bisbenzamidines 66–73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin and thrombin inhibition were observed.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.     

Biodegradation of cellulosic materials: Substrates,microorganisms, enzymes and products

Cellulosic materials are the only renewable resources available in large quantities which need to be properly utilized to meet our needs of energy, chemicals, food and feed for a long-range solution. A variety of lignocellulosic materials are available and microorganisms capable of degrading either one or more of the three main constituents, viz. cellulose, hemicellulose and lignin have been studied. At least three different enzymes of the multicomponent cellulase system, i.e. cellobiohydrolase endo-glucanase and β-glucosidase are involved in the degradation of crystalline cellulose into glucose. Their mode of action and the manner in which they bring about hydrolysis of crystalline cellulose is discussed in detail. The involvement of parallel enzymes for hemicellulose degradation is also known to some extent but needs to be studied more elaborately, independently and in combination with cellulases. The potential of cellulosic biomass as a source of fuel and petroleum-sparing substances is also reviewed.     

Organization of the gene for batroxobin, a thrombin-like snake venom enzyme. Homology with the trypsin/kallikrein gene family

We have isolated and analyzed the gene for batroxobin, a thrombin-like snake venom enzyme. Three overlapping DNA segments containing the entire batroxobin gene were identified. Sequence analysis revealed that the batroxobin gene spans 8 kilobase pairs and contains five exons. Mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of batroxobin, His-41, Asp-86, and Ser-178, are encoded by separate exons, exons 2, 3, and 5, respectively. The exon/intron organization of the batroxobin gene is different from that of the prothrombin gene but very similar to those of the trypsin and kallikrein genes. These results indicate that batroxobin is not a member of the prothrombin family but one of the trypsin/kallikrein family. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.     

A new beta-naphthylamide substrate of p-guanidino-L-phenylalanine for trypsin and related enzymes

N alpha-Benzyloxycarbonyl-p-guanidino-L-phenylalanine beta-naphthylamide (Z-GPA-beta NA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of N alpha-benzyloxycarbonyl-L-arginine beta-naphthylamide (Z-Arg-beta NA). Both Z-GPA-beta NA and Z-Arg-beta NA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-beta NA was hydrolyzed slowly by thrombin, while Z-GPA-beta NA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-beta NA by papain was slower than that of Z-Arg-beta NA. Neither beta-naphthylamide substrate was hydrolyzed by alpha-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-beta NA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-beta NA. Contributions of the benzene ring in the side chain of Z-GPA-beta NA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two beta-naphthylamide substrates. Z-GPA-beta NA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.     

Geometry of binding of the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino phenylalanine to thrombin and trypsin. X-ray crystal structures of their trypsin complexes and modeling of their thrombin complexes

The X-ray crystal structures of the complexes formed with bovine trypsin and the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino-D,L-phenylalanine (3-TAPAP, 4-TAPAP and 4-TGPAP) were determined with data to 1.8 A resolution. The L-stereoisomer of 3-TAPAP binds as a compact entity into the active site of trypsin, with the amidino and the carbonyl groups of the central amidinophenylalanyl residue hydrogen-bonded to Gly216 of trypsin. According to modeling and energy minimization, 3-TAPAP fits perfectly in this conformation to the more restrictive thrombin active site also (Bajusz et al. (1978) Int. J. Pept. Prot. Res. 12, 217-221); the piperidine moiety extends into the cage-like S2 subsite of thrombin, but leaves room for additional substituents which might help to improve binding and pharmacological properties. In contrast, 4-TAPAP and 4-TGPAP bind only weakly and in an extended conformation to trypsin; their considerably enhanced affinities for thrombin would suggest a more compact binding to thrombin.