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1.
T Komiyama T L Bigler N Yoshida K Noda M Laskowski 《The Journal of biological chemistry》1991,266(17):10727-10730
Turkey ovomucoid third domain with P1 Leu18 at its reactive site is an excellent inhibitor of chymotrypsin and elastase and of many other serine proteinases with related specificities. Semisynthetic replacement of P1 Leu18 by Lys18 causes the expected change into a trypsin inhibitor. Strikingly, semisynthetic replacement P1 Leu18 to Glu18 changes turkey ovomucoid third domain into a powerful inhibitor of Glu-specific Streptomyces griseus proteinase, GluSGP. Of the 131 natural avian ovomucoid third domains we have sequenced none have P1 Glu18, but several avian ovomucoid first domains have P1 Glu24. They are weak to moderate inhibitors of GluSGP. 相似文献
2.
A M Andrianov 《Molekuliarnaia biologiia》1991,25(5):1215-1225
The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters. 相似文献
3.
Effect of single amino acid replacements on the thermodynamics of the reactive site peptide bond hydrolysis in ovomucoid third domain 总被引:4,自引:0,他引:4
We have measured equilibrium constants, Khyd, at pN 6 for the hydrolysis of the reactive site peptide bond (bond between residues 18 and 19) in 42 sequenced variants (39 natural, 3 semisynthetic) of avian ovomucoid third domains. The values range from 0.4 to approximately 35. In 35 cases the effect of a single amino acid replacement on Khyd could be calculated, 13 are without effect and 22 range from a factor of 1.25 to 5.5. Several, but not all, of the effects can be rationalized in terms of residue-residue interactions that are affected by the reactive site hydrolysis. As the measurements are very precise it appears that additional measurements on designed rather than natural variants should allow for the precise measurement of side-chain--side-chain interaction energies. 相似文献
4.
Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain
The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues. 相似文献
5.
We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor. Turkey ovomucoid third domain was converted to modified (the reactive site peptide bond hydrolyzed) form as documented by sequencing. Complexes of all eight enzymes both with virgin and with modified inhibitor were prepared. All 16 complexes were subjected to kinetically controlled dissociation, and all 16 produced predominantly virgin (greater than 90%) inhibitor, thus proving our point. During this investigation, we found that both alpha-chymotrypsin and especially S. griseus proteinase B convert virgin to modified turkey ovomucoid third domain, even in the pH range 1-2, a much lower pH than we expected. We have also measured rate constants kon and kon* for the association of virgin and modified turkey ovomucoid third domain with several serine proteinases. The kon/kon* ratio is 4.8 X 10(6) for chymotrypsin, but it is only 1.5 for subtilisin Carlsberg. A number of generalizations concerning reactive sites of protein proteinase inhibitor are proposed and discussed. 相似文献
6.
Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues. 总被引:3,自引:3,他引:0
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T. L. Bigler W. Lu S. J. Park M. Tashiro M. Wieczorek R. Wynn M. Laskowski Jr 《Protein science : a publication of the Protein Society》1993,2(5):786-799
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. 相似文献
7.
Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues 总被引:12,自引:0,他引:12
M Laskowski I Kato W Ardelt J Cook A Denton M W Empie W J Kohr S J Park K Parks B L Schatzley 《Biochemistry》1987,26(1):202-221
Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron. 相似文献
8.
The solution structure of modified turkey ovomucoid third domain (OMTKY3*) was investigated by high-resolution proton NMR techniques. OMTKY3* was obtained by enzymatic hydrolysis of the scissile reactive site peptide bond (Leu18-Glu19) in turkey ovomucoid third domain (OMTKY3). All of the backbone proton resonances were assigned to sequence-specific residues except the NH's of Leu1 and Glu19, which were not observed. Over 80% of the side-chain protons also were assigned. The secondary structure of OMTKY3*, as determined from assigned NOESY cross-peaks and identification of slowly exchanging amide protons, contains antiparallel beta-sheet consisting of three strands (residues 21-25, 28-32, and 49-54), one alpha-helix (residues 33-44), and one reverse turn (residues 26-28). This secondary structure closely resembles that of OMTKY3 in solution [Robertson, A. D., Westler, W. M., & Markley, J. L. (1988) Biochemistry (preceding paper in this issue)]. On the other hand, changes in the tertiary structure of the protein near to and remote from the cleavage site are indicated by differences in the chemical shifts of numerous backbone protons of OMTKY3 and OMTKY3*. 相似文献
9.
What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? 总被引:14,自引:0,他引:14
Proteinases perform many beneficial functions that are essential to life, but they are also dangerous and must be controlled. Here we focus on one of the control mechanisms: the ubiquitous presence of protein proteinase inhibitors. We deal only with a subset of these: the standard mechanism, canonical protein inhibitors of serine proteinases. Each of the inhibitory domains of such inhibitors has one reactive site peptide bond, which serves all the cognate enzymes as a substrate. The reactive site peptide bond is in a combining loop which has an identical conformation in all inhibitors and in all enzyme-inhibitor complexes. There are at least 18 families of such inhibitors. They all share the conformation of the combining loops but each has its own global three-dimensional structure. Many three-dimensional structures of enzyme-inhibitor complexes were determined. They are frequently used to predict the conformation of substrates in very short-lived enzyme-substrate transition state complexes. Turkey ovomucoid third domain and eglin c have a Leu residue at P(1). In complexes with chymotrypsin, these P(1) Leu residues assume the same conformation. The relative free energies of binding of P(1) Leu (relative to either P(1) Gly or P(1) Ala) are within experimental error, the same for complexes of turkey ovomucoid third domain, eglin c, P(1) Leu variant of bovine pancreatic trypsin inhibitor and of a substrate with chymotrypsin. Therefore, the P(1) Leu conformation in transition state complexes is predictable. In contrast, the conformation of P(1) Lys(+) is strikingly different in the complexes of Lys(18) turkey ovomucoid third domain and of bovine pancreatic trypsin inhibitor with chymotrypsin. The relative free energies of binding are also quite different. Yet, the relative free energies of binding are nearly identical for Lys(+) in turkey ovomucoid third domain and in a substrate, thus allowing us to know the structure of the latter. Similar reasoning is applied to a few other systems. 相似文献
10.
Cytotoxic and Apoptosis‐Inducing Activities,and Anti‐Tumor‐Promoting Effects of Cyanogenated and Oxygenated Triterpenes
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Takashi Kikuchi Kenta Ishii Eri Ogihara Jie Zhang Motohiko Ukiya Harukuni Tokuda Takashi Iida Reiko Tanaka Toshihiro Akihisa 《化学与生物多样性》2014,11(4):491-504
Two of each semisynthetic lanostane‐ and cycloartane‐type triterpenes with a cyano‐enone functionality, i.e., 13 and 18 , and 23 and 28 , respectively, sixteen of their synthetic intermediates, 9 – 12, 14 – 17, 19 – 22 , and 24 – 27 , along with seven semisynthetic oxygenated triterpene acetates, 29 – 35 , and eight natural hydroxy triterpenes, 1 – 8 , were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines. One natural triterpene, 8 , and ten semisynthetic triterpenes, 9, 13, 15, 18, 23, 25, 28, 29, 32 , and 33 , exhibited potent cytotoxicities against one or more cell lines with IC50 values in the range of 1.4–9.9 μM . Two lanostane‐type triterpenes with a cyano‐enone functionality, 3‐oxolanosta‐1,8,24‐triene‐2‐carbonitrile ( 13 ) and 3‐oxolanosta‐1,8‐diene‐2‐carbonitrile ( 18 ), induced apoptosis in HL60 cells, as observed by membrane phospholipid exposure in flow cytometry. Western blot analysis showed that 13 and 18 significantly reduced procaspases‐3, ‐8, and ‐9, and increased cleaved caspases‐3, ‐8, and ‐9. These findings indicated that compounds 13 and 18 induced apoptosis in HL60 cells via both the mitochondrial and the death receptor‐mediated pathways. In addition, upon evaluation of the inhibitory effects on Epstein? Barr virus early antigen (EBV‐EA) activation induced with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells, seven natural triterpenes, 1 – 6 and 8 , and ten semisynthetic triterpenes, 9, 10, 14, 15, 19, 20, 24, 25, 29 , and 30 , exhibited inhibitory effects which were higher than that of β‐carotene, a vitamin A precursor studied widely in cancer‐chemoprevention animal models. 相似文献
11.
A biologically active semisynthetic hybrid of horse heart cytochrome c has been prepared by combining the heme peptide 1 through 65 (HP 1-65), prepared by CNBr cleavage of natural cytochrome c, with a semisynthetic peptide corresponding to positions 66 through 104. A fully protected synthetic peptide 66--79 was prepared by a modified solid phase peptide synthesis procedure and was converted to its N-hydroxysuccinimide ester. A peptide corresponding to residues 81--104 of cytochrome c was also isolated from the CNBr cleavage mixture and its epsilon-amino groups and tyrosyl hydroxyl group were protected selectively with the t-butyloxycarbonyl group. This partially protected peptide was reacted with t-butyloxycarbonyl methionine N-hydroxysuccinimide ester to give a derivative having methionine at position 80. This product was deprotected, purified and then t-butyloxycarbonyl groups were again introduced specifically on the epsilon-amino groups to give the peptide, Boc(Lys,Tyr)80--104. A semisynthetic peptide corresponding to residues 66 through 104 of cytochrome c was prepared by condensing the synthetic peptide 66--79 N-hydroxysuccinimide ester with t-butyloxycarbonyl (Lys,Tyr)80--104. The semisynthetic product was deprotected, purified and combined under anaerobic conditions with a heme peptide, HP 1-65, that was isolated from the products of CNBr cleavage of native cytochrome c. The reconstituted semisynthetic cytochrome c was purified by ion exchange chromatography and was shown to have the same oxygen uptake as native cytochrome c when assayed in the succinate oxidase system. 相似文献
12.
B E Kemp 《The Journal of biological chemistry》1979,254(8):2638-2642
A series of synthetic peptide analogs of the cardiac troponin inhibitory subunit (TN-1) phosphorylation site sequence, Arg12-Pro-Ala-Pro-Ala-Val-Arg18-Arg19-Ser20-Asp21-Arg22-Ala, have been tested as substrates for the catalytic subunit of the cyclic AMP-dependent protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase). As substrates, these peptides were generally inferior to the pyruvate kinase analog peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly or its COOH-terminal amide analog. Replacing Arg-19 with alanine had only a minor effect on the kinetics of phosphorylation of the TN-1 peptide analog. In contrast, replacement of Arg-22 and Arg-18 with alanine resulted in marked enhancement and reduction of the Vmax, respectively. The results of this study have demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase. In the case of the TN-1 peptide analogs, the contribution of the 4 arginine residues can be distinguished in terms of their influence on the kinetics of phosphorylation. 相似文献
13.
Hilpert K Wessner H Schneider-Mergener J Welfle K Misselwitz R Welfle H Hocke AC Hippenstiel S Höhne W 《The Journal of biological chemistry》2003,278(27):24986-24993
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase. 相似文献
14.
Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure. 相似文献
15.
We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1-M6), four peptide fragments, M12 (1-52), M123 (1-73), M1234 (1-88) and M12345 (1-98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1-98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1-98) with M56 (89-110) showed weak but distinct RNase activity. Unfolded M12345 (1-96) was constructed by removal of two residues from the C-terminus of M12345 (1-98). The mixture of M12345 (1-96) with M56 (89-110) also showed RNase activity. Further, the interaction endowed M12345 (1-96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution. 相似文献
16.
J G Beeley 《The Biochemical journal》1976,155(2):345-351
Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule. 相似文献
17.
The substrate-like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor-proteinase interactions of the standard type. 1H,15N and 13C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3)2 and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15N and 13C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13-P22 and N33-A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase-contact residues of OMTKY3 defined previously on the basis of X-ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through-bond 15N-13C correlations between P1-13C' and P1'-15N in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate. 相似文献
18.
T J Palker R M Scearce T D Copeland S Oroszlan B F Haynes 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(7):2393-2397
To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific. 相似文献
19.
Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C. 总被引:1,自引:0,他引:1
We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site. 相似文献
20.
1. Aspergillopeptidase B rapidly hydrolyses the -Leu18-Glu19-reactive site peptide bond in turkey ovomucoid third domain (OMTKY3) within the pH-range of 4.0-8.4. The reaction proceeds to equilibrium between OMTKY3 and its modified form with the reactive site peptide bond cleaved (OMTKY3). 2. The dependence of the equilibrium constant (Khyd) on pH indicates that hydrolysis of the reactive site peptide bond apparently does not perturb the pK-values of any preexistent ionizable groups in OMTKY3. 3. The obtained Khyd0 value indicates that free energies of OMTKY3 and OMTKY3 are essentially the same. 4. Hydrolysis of the reactive site peptide bond by aspergillopeptidase B at neutral pH is about 60 times faster than the same reaction catalyzed by subtilisin (Carlsberg), the enzyme strongly inhibited by OMTKY3. 5. Resynthesis of the reactive site peptide bond at neutral pH catalyzed by aspergillopeptidase B (reverse reaction) is almost four orders of magnitude faster than the forward reaction. 相似文献