Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds

Preparations of low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat (Fagopyrum esculentum) seeds by chromatography of seed extract on trypsin-Sepharose 4B, Mono-Q, and Mono-S ion exchangers (FPLC regime). Their molecular masses, determined by mass spectrometry, were 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c), and 6031 daltons (BWI-4c). All of the inhibitors possess high pH- and thermal stability in the pH range 2-12. In addition to trypsin, BWI-3c and BWI-4c inhibited chymotrypsin and subtilisin-like bacterial proteases. The N-terminal sequences of all of the inhibitors were determined: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues), and BWI-4c (20 residues). In their physicochemical properties and N-terminal amino acid sequences, the buckwheat seed trypsin inhibitors BWI-3c and BWI-4c appear to belong to potato proteinase inhibitor I family.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

Proteinase inhibitors from the tropical sea anemone Radianthus macrodactylus: Isolation and characteristic

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.     

Characterization of midgut proteinase activities of white grubs: Lepidiota noxia,Lepidiota negatoria,and Antitrogus consanguineus (scarabaeidae,melolonthini)

The proteinases in the midguts of three scarab white grub species, Lepidiota noxia, L. negatoria, and Antitrogus consanguineus, were investigated to classify the proteinases present and to determine the most effective proteinase inhibitor for potential use as an insect control agent. pH activity profiles indicated the presence of serine proteinases and the absence of cysteine proteinases. This was confirmed by the lack of inhibition by specific cysteine proteinase inhibitors. Trypsin, chymotrypsin, elastase, and leucine aminopeptidase activities were detected by using specific synthetic substrates. A screen of 32 proteinase inhibitors produced 9 inhibitors of trypsin, chymotrypsin, and elastase which reduced proteolytic activity by greater than 75%. © 1995 Wiley-Liss, Inc.     

Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.     

Serine Proteinase Inhibitors from Eggs and Larvae of Tick Boophilus microplus: Purification and Biochemical Characterization

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as E1, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCl buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant K _i determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTIs) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The K _i for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.     

Trypsin isoinhibitors of lucerne: Association with leaf fraction 1 protein

Trypsin inhibitory activity occurring in the vegetative portion of lucerne (Medicago sativa L.) comprised two heat-labile isoinhibitors with isoele     

Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.     

丝瓜籽中一种具有翻译抑制活性和胰蛋白酶抑制剂活性的多肽——Luffin P1的纯化和性质

通过硫酸铵分级沉淀、CM 5 2阳离子交换层析、蓝胶亲和层析和FPLCMonoS阳离子交换层析 ,从丝瓜籽抽提液中分离到一种多肽luffinP1。经MALDI TOFMS测得其分子量为 5 2 2 6 .5。氨基酸序列测定及同源性分析发现 ,luffinP1的N端 11个氨基酸序列与丝瓜籽中的一种 6 .5K富含Arg Glu的多肽AGRP的部分序列相同 ,并与南瓜籽中一种胰蛋白酶抑制剂C2肽具有很高的同源性。体外分析表明 ,luffinP1同时具有两种生物活性 :(1)对兔网织红细胞裂解液系统蛋白质生物合成有较强的抑制作用 ,IC50 为 0 .6nmol L ;(2 )具有明显的胰蛋白酶抑制活性 ,IC50 为 2 2 μmol L。     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Prokaryotic DNA Methyltransferases: The Structure and the Mechanism of Interaction with DNA

The review considers current views on the function of DNA methyltransferases (MTases) that belong to prokaryotic type II restriction–modification systems. A commonly accepted classification of MTases is described along with their primary and tertiary structures and molecular mechanisms of their specific interaction with DNA (including methylation). MTase inhibitors are also considered. Special emphasis is placed on the flipping of the target heterocyclic base out of the double helix and on the methods employed in its analysis. Base flipping is a fundamentally new type of DNA conformational changes and is also of importance in the case of other DNA-operating enzymes. MTases show unique sequence homology, and are similar in structure of functional centers and in the mechanism of methylation. These data contribute to the understanding of the general biological significance of methylation, since prokaryotic and eukaryotic MTases are structurally and functionally similar.     

Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na₂SO₄) and the anionic buffer 2-( N -morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent K _m of the enzyme for the cofactor 6,7-dimethyltetrahydropterin (DMPH₄) and an increase in the V _max for tyrosine and DMPH₄. The K _m for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3-( N -morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N -(2-acetamido)-2-amino-ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.