Structural consequences of the natural substitution, E9K, on reactive-site-hydrolyzed squash (Cucurbita maxima) trypsin inhibitor (CMTI), as studied by two-dimensional NMR.

Sequence-specific hydrogen-1 NMR assignments were made to all of the 29 amino acid residues of reactive-site-hydrolyzed Cucurbita maxima trypsin inhibitor I (CMTI-I*) by the application of two-dimensional NMR (2D NMR) techniques, and its secondary structural elements (two tight turns, a 3(10)-helix, and a triple-stranded beta-sheet) were identified on the basis of short-range NOESY cross peaks and deuterium-exchange kinetics. These secondary structural elements are present in the intact inhibitor [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] and are unaffected by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6, in accordance with the earlier conclusion reached for CMTI-III* [Krishnamoorthi, R., Gong, Y.-X., Lin, C. S., & VanderVelde, D. (1992) Biochemistry 31, 898-904]. Chemical shifts of backbone hydrogen atoms, peptide NH's, and C alpha H's, of CMTI-I* were compared with those of the intact inhibitor, CMTI-I, and of the reactive-site-hydrolyzed, natural, E9K variant, CMTI-III*. Cleavage of the Arg5-Ile6 peptide bond resulted in changes of chemical shifts of most of the backbone atoms of CMTI-I, in agreement with the earlier results obtained for CMTI-III. Comparison of chemical shifts of backbone hydrogen atoms of CMTI-I* and CMTI-III* revealed no changes, except for residues Glu9 and His25. However, the intact forms of the same two proteins, CMTI-I and CMTI-III, showed small but significant perturbations of chemical shifts of residues that made up the secondary structural elements of the inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis of new trypsin, chymotrypsin and elastase inhibitors by amino-acid substitutions in a trypsin inhibitor from squash seeds (CMTI III)

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.     

The serine proteinase inhibitor from summer squash (Cucurbita pepo): some structural features, stability and proteolytic degradation

The serine proteinase inhibitor from summer squash seeds (CPTI-II) with Mr of about 3250 contains three disulphide bridges and is unusually resistant to denaturing agents (e.g. 10% trichloroacetic acid at about 100 degrees C), thermolysin and proteinase V8 from Staphylococcus aureus. The inhibitor is digested by pepsin; the digestion of the virgin form proceeds more rapidly than when the peptide bond of the reactive site is broken. The inhibitor is not specifically reduced by sodium borohydride at pH 8.8, and almost full reactivation of the inhibitor reduced by dithiothreitol takes place at pH 8.15 in the presence of EDTA and the reduced + oxidized glutathione system. The inhibitor was crystallized from methanol. CD spectra point to the occurrence of beta-turns in the secondary structure of the inhibitor.     

Synthesis of an elastase inhibitor by monosubstitution of arginine-5 with valine at the reactive site in a trypsin inhibitor from squash seeds (CMTI III)

Using the solid-phase procedure an analog of trypsin inhibitor CMTI III containing Val5 instead of Arg5 in position P1, was synthesized. The substitution in only this one position P1 increased the affinity of synthetic inhibitor to porcine pancreatic elastase and human leukocyte elastase by the factor of 10(3) and 10(7), respectively.     

Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III)

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.     

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Purification,properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.     

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.     

Kinetic mechanism and order of substrate binding for sn-glycerol-3-phosphate acyltransferase from squash (Cucurbita moschata)

sn-Glycerol-3-phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three-dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451-453; Turnbull et al. (2001) Structure 9, 347-353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl-acyl carrier protein (acyl-ACP) substrates. The reaction proceeds via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate. We have also determined that the reaction will proceed with C(4:0)-CoA, C(6:0)-CoA and C(12:0)-ACP substrates, allowing a wider choice of acyl groups for future co-crystallisation studies.     

Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.     

A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.     

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties

Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.     

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of three inhibitors

Three trypsin inhibitors were isolated from summer squash (Cucurbita pepo) seeds and purified to homogeneity by fractionation with ammonium sulphate and methanol, ion-exchange chromatography and gel filtration. All three inhibitors have lysine at their active site. Two of them (II, IV) show the same isoelectric point (at pH 5.6), amino acid composition and molecular mass (3259). The third inhibitor (III) of molecular mass of 3654 and isoelectric point at 4.9 has additionally one histidine residue and two glutamic acid residues more per molecule.     

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.     

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.     

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.     

Ribokinase from E. coli: expression, purification, and substrate specificity

Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported.