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I P Kuranova - One of the best experts on this subject based on the ideXlab platform.
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structure of the complex of <B>CarBoxypeptidaseB> B and n sulfamoyl l arginine
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015Co-Authors: V K Akparov, Nikolay Sokolenko, V I Timofeev, I P KuranovaAbstract:: Porcine pancreatic <B>CarBoxypeptidaseB> B (EC 3.4.23.6) was complexed with a staBle transition-state analogue, N-sulfamoyl-L-arginine, in which an S atom imitates the sp(3)-hyBridized carBon in the scissile-Bond surrogate. Crystals were grown in a form Belonging to the same space group, P41212, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 A. The molecule was refined and the positions of non-H atoms of the inhiBitor and water molecules were defined using difference Fourier maps. The enzyme-inhiBitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation Between the complexed and uncomplexed forms of <B>CarBoxypeptidaseB> B are shown. The inhiBitor is Bound in a curved conformation in the active-site cleft, and the sulfamide group is Bound to the Zn ion in an asymmetric Bidentate fashion. The complex is staBilized By hydrogen Bonds Between the N1/N2 guanidine group of the inhiBitor and the Asp255 carBoxyl of the enzyme. The side-chain CH2 groups of the inhiBitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may Bind to <B>CarBoxypeptidaseB> B and therefore provides an insight into the structural Basis of <B>CarBoxypeptidaseB> B selectivity, which is useful for the rational design of a <B>CarBoxypeptidaseB> with improved selectivity for industrial recomBinant pro-insulin processing.
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Crystallization and preliminary X-ray diffraction study of porcine <B>CarBoxypeptidaseB> B
Crystallography Reports, 2015Co-Authors: V. Kh. Akparov, V I Timofeev, I P KuranovaAbstract:Crystals of porcine pancreatic <B>CarBoxypeptidaseB> B have Been grown in microgravity By the capillary counter-diffusion method through a gel layer. The X-ray diffraction study showed that the crystals Belong to sp. gr. P 4_12_12 and have the following unit-cell parameters: a = B = 79.58 Å, c = 100.51 Å; α = β = γ = 90.00°. The X-ray diffraction data set suitaBle for the determination of the three-dimensional structure at atomic resolution was collected from one of the grown crystals at the SPring 8 synchrotron facility to 0.98 Å resolution.
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preparation crystallization and preliminary x ray diffraction study of mutant <B>CarBoxypeptidaseB> t containing the primary specificity pocket of <B>CarBoxypeptidaseB> B
Crystallography Reports, 2010Co-Authors: Kh V Akparov, V I Timofeev, A M Grishin, I P KuranovaAbstract:RecomBinant G215S, A251G, T257A, D260G, T262D mutant <B>CarBoxypeptidaseB> T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated By X-ray diffraction. RecomBinant mutant <B>CarBoxypeptidaseB> T containing the primary specificity suBsite compositionally identical to that of pancreatic <B>CarBoxypeptidaseB> B crystallizes in the same space group as the natural enzyme. The crystals Belong to sp. gr. P6322; the unit-cell parameters are a = B = 157.867 A, c = 104.304 A, α = β = 90°, γ = 120°. X-ray diffraction data suitaBle for determining the three-dimensional structure at atomic resolution were collected from one crystal.
Yuan Qin-sheng - One of the best experts on this subject based on the ideXlab platform.
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Research on RecomBinant <B>CarBoxypeptidaseB> B and SoluBility of Its Inclusion Body
Food and Drug, 2007Co-Authors: Yuan Qin-shengAbstract:OBjective The expression plasmid and strain of recomBinant <B>CarBoxypeptidaseB> B (rCPB) were constructed. The soluBility of inclusion Body of the expressed <B>CarBoxypeptidaseB> (CPB) was also studied. Methods The recomBinant plasmid of CPB, pET-21a-CPB, was constructed and transformed into E. coli BL21(DE3). The expression was induced in 25℃and 37℃, respectively. The oBtained inclusion Bodies were dissolved in different denaturing solution systems. The protein concentration was determined in order to indicate the soluBility of CPB inclusion Bodies in the denaturing solution. And proBaBle mechanism for the soluBility differences was illuminated By the analysis of unreduced SDS-PAGE. Results The purity and following treatment of inclusion Bodies of CPB were influenced By the culturing temperature after the inducement with IPTG. The soluBility of inclusion Bodies of rCPB in 10mol/L urea was 2￣3 times higher than in 8 mol/L urea, and the renature solution with 0.75%β-ME in 10mol/L urea could increase the soluBility of inclusion Bodies of rCPB, comparing with that in 10mol/L urea simply. As a result of the analysis of unreduced SDS-PAGE, the content of large molecular rCPB polymers was decreased inβ-ME solutions as well. Conclusion The rCPB can Be expressed successfully and the soluBility of its inclusion Bodies can Be increased By experiment.
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Aduances in <B>CarBoxypeptidaseB> B
Pharmaceutical biotechnology, 2006Co-Authors: Yuan Qin-shengAbstract:<B>CarBoxypeptidaseB> B is a necessary enzyme especially in the processing of recomBinant insulin and is often widely used in the sequencing of proteins.In this article,the structure,method of activity deter mination,characteristics,staBility,the storage condition,the resolution ways,the production and properties of recomBinant <B>CarBoxypeptidaseB> B were all summarized.
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SoluBle Expression of RecomBinant Pro-<B>CarBoxypeptidaseB> B in E.coli and Purification of Active <B>CarBoxypeptidaseB> B
Pharmaceutical biotechnology, 2006Co-Authors: Yuan Qin-shengAbstract:The expression condition of recomBinant pCPB was optimized to achieve the soluBle expression in E.coli;the recomBinant pCPB was enzymatically cleaved and suBsequently purified to oBtain active <B>CarBoxypeptidaseB> B.Through adjusting the inducing temperature,inducing time,and inducer concentration,pCPB was expressed in supernatant in E.coli;the special activity of CPB was increased By improving the enzymatic cleavage condition.The rude CPB was purified By DEAE-FF chromatography and Octyl-FF chromatography.The highest soluBle expression level of recomBinant pCPB was oBtained after Being induced at 15℃ for 20()h and the concentration of IPTG was 0.1()mmol/L.The active CPB was purified suBsequently By DEAE-FF and Octyl-FF chromatography to oBtain the active <B>CarBoxypeptidaseB> B with special activity aBout 128.2()u/mg protein,and total activity recovery was 39%.The soluBle expression of recomBinant pro<B>CarBoxypeptidaseB> B was achieved in E.coli.We got the active CPB after enzymatical cleavage and purification.
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Renaturation of RecomBinant Pro-<B>CarBoxypeptidaseB> B By Gel Filtration and Purifi cation of Active <B>CarBoxypeptidaseB> B
Chinese Journal of Biologicals, 2005Co-Authors: Yuan Qin-shengAbstract:OBjective To develop the methods for renatura tion of recomBinant pro-<B>CarBoxypeptidaseB> B and for purification of active carBoxype ptidase B.Methods After recomBinant E.coli was cultured By fermentation,the expressed pro-<B>CarBoxypeptidaseB> B,in a form of inclusion Body,w as washed and denaturalized,then purified and re-naturalized By gel filtration. The oBtained solution was digested with trypsin,from which <B>CarBoxypeptidaseB> B wa s purified By DEAE-FF anion exchange chromatography.The purity of carBoxypeptid ase B was determined By SDS-PAGE.Results In case of the same f inal concentration of pro-caroxypeptidase B,the renaturation efficiency By gel filtration increased By 50% compared with that By dilution.After purification By DEAE-FF anion exchange chromatography,the purity of <B>CarBoxypeptidaseB> reached a Bove 95%,and its specific activity determined using Hippuryl-L-arg as a suBstr ate was 150 u/mg.Conclusion The developed methods were effectiv e and laid a foundation of further study on property of caroxypeptidase. [
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Research on denatutation and renaturation of recomBinant pro-<B>CarBoxypeptidaseB> B expressed from Escherichia coli
Journal of Molecular Science, 2005Co-Authors: Yuan Qin-shengAbstract:RecomcomBinant pro-<B>CarBoxypeptidaseB> B formed insoluBle inclusion Bodies when overexpressed in Escherichia coli,and it must Be denatured and renatured in vitro Before it acquired activity. Various Buffers including denaturants, extreme pH, detergents and Tris Buffer at high temperature were used to soluBilize inclusion Bodies and pH =11 was proved to have good soluBility. To increase the renaturation yield of denatured pro-<B>CarBoxypeptidaseB> B,the Basic renaturation conditions of pro-<B>CarBoxypeptidaseB> B were decided through qualitative and quantitative analysis of denaturant concentration, pH,temperature, the ratio of reduced and oxidized reagent and dilution ways. The optimized renaturation Buffer was as follows: 20 mmol/L Tris-HCl, pH = 9. 5, 150 μg/mL Pro-CPB concentration,1 mmol/L GSH,0. 5 mol/L urea. The renaturation effect was three times than Before.
Francesc X Aviles - One of the best experts on this subject based on the ideXlab platform.
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Expression, purification and characterization of porcine pancreatic <B>CarBoxypeptidaseB> B from Pichia pastoris for the conversion of recomBinant human insulin
Enzyme and Microbial Technology, 2007Co-Authors: Manuel Mansur, Liliam Martínez, Mariela Pérez, Maday Alonso-del-rivero, Isvaloy Márquez, Yanay Proenza, Laura Varas, Francesc X AvilesAbstract:ABstract Pancreatic or tissue <B>CarBoxypeptidaseB> B (CPB), a key enzyme involved in insulin conversion and highly specific for excising C-terminal Lys and Arg residues from peptides and proteins, was expressed at high level and purified from a recomBinant Pichia pastoris strain. A cDNA containing the porcine pancreatic pro-<B>CarBoxypeptidaseB> B (proCPB) fused to the Saccharomyces cerevisiae alpha factor secretion signal was cloned into the pPIC3K vector under control of P. pastoris AOX1 promoter. After 72 h of growth on methanol, proCPB accumulated until 320 mg L −1 , representing 70% of total proteins in culture supernatant. A single stepwise ion exchange purification process with Q-Sepharose at increasing concentrations of ammonium acetate allowed recovery of 65% proCPB in a single fraction. The dialyzed protein was activated with trypsin and its activity was tested with the synthetic suBstrate Hippuryl- l -Arg. The kinetic parameters K M and V max , as well as inhiBition constant K i against a specific inhiBitor were calculated and found similar to those of the wild-type enzyme. The enzyme efficiently removed amino acids Lys and Arg from the spacer of an insulin precursor (B 1–30 -LysArg-A 1–21 ) expressed in yeast and previously cleaved with trypsin. The enzyme was found staBle for 4 h at pH 11.8, a useful property for performing Both enzyme reactions with trypsin and CPB in a single step.
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Response of the digestive system of Helicoverpa zea to ingestion of potato <B>CarBoxypeptidaseB> inhiBitor and characterization of an uninhiBited <B>CarBoxypeptidaseB> B.
Insect Biochemistry and Molecular Biology, 2006Co-Authors: Àlex Bayés, Josep Vendrell, Monica Rodriguez De La Vega, Francesc X Aviles, Maarten Anthonie Jongsma, Jules BeekwilderAbstract:<B>CarBoxypeptidaseB> activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different <B>CarBoxypeptidaseB>s in lepidopteran protein digestion, the effect of potato <B>CarBoxypeptidaseB> inhiBitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of SoyBean Kunitz Trypsin InhiBitor. Analysis of <B>CarBoxypeptidaseB> activity in the guts showed that ingested PCI remained active in the gut, and completely inhiBited the activity of <B>CarBoxypeptidaseB>s A and O. Interestingly, <B>CarBoxypeptidaseB> B activity was not affected By PCI. All previously descriBed enzymes from the same family, Both from insect or mammalian origin, have Been found to Be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of <B>CarBoxypeptidaseB> B activity resistant to PCI in most of them. The H. zea <B>CarBoxypeptidaseB> B enzyme (CPBHz) was purified from gut content By affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recomBinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The suBstrate specificity of recomBinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to Be highly selective for C-terminal lysine residues. InhiBition By PCI appeared to Be pH-dependent.
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244 – <B>CarBoxypeptidaseB> B
Handbook of Proteolytic Enzymes, 2004Co-Authors: Francesc X AvilesAbstract:PuBlisher Summary This chapter descriBes the activity, specificity and structural chemistry of <B>CarBoxypeptidaseB> B. <B>CarBoxypeptidaseB> B is highly specific for excising C-terminal Lys and Arg residues from peptides and proteins with a preference for arginine. It also acts at a slower rate on C-terminal Val, Leu, He, Asn, Gly or Gin. A preference for Ala in position P2 of synthetic suBstrates has Been oBserved. Typical assay methods use Bz-Gly-f Arg or FA-Ala┼Arg as suBstrates. Assays are carried out at pH 7.5–7.8 and 0.1–0.15 M sodium chloride. A numBer of synthetic inhiBitors of Basic <B>CarBoxypeptidaseB>s are termed ‘Biproduct analogs.’ Guanidinoethylmercaptosuccinic acid and 2-mercaptomethyl-3-guanidinoethyl-propanoic acid are relevant examples. Other powerful, naturally occurring proteinaceous inhiBitors are potato <B>CarBoxypeptidaseB> inhiBitor and leech <B>CarBoxypeptidaseB> inhiBitor, Both of which have K values in the nanomolar range. <B>CarBoxypeptidaseB> B has Been isolated from the pancreas of several species including human , cattle , pig , ostrich and others, and human serum and longitudinal muscle layer of cattle small intestine . A staBle enzyme can Be purified from pancreatic acetone powder. Internal proteolysis and degradation may occur if the selected source is pancreatic secretion. The purified enzyme is staBle for years in the precipitated or the frozen states.
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244 <B>CarBoxypeptidaseB> B
Handbook of Proteolytic Enzymes (Second Edition)#R##N#Aspartic and Metallo Peptidases, 2004Co-Authors: Francesc X AvilesAbstract:PuBlisher Summary This chapter descriBes the activity, specificity and structural chemistry of <B>CarBoxypeptidaseB> B. <B>CarBoxypeptidaseB> B is highly specific for excising C-terminal Lys and Arg residues from peptides and proteins with a preference for arginine. It also acts at a slower rate on C-terminal Val, Leu, He, Asn, Gly or Gin. A preference for Ala in position P2 of synthetic suBstrates has Been oBserved. Typical assay methods use Bz-Gly-f Arg or FA-Ala┼Arg as suBstrates. Assays are carried out at pH 7.5–7.8 and 0.1–0.15 M sodium chloride. A numBer of synthetic inhiBitors of Basic <B>CarBoxypeptidaseB>s are termed ‘Biproduct analogs.’ Guanidinoethylmercaptosuccinic acid and 2-mercaptomethyl-3-guanidinoethyl-propanoic acid are relevant examples. Other powerful, naturally occurring proteinaceous inhiBitors are potato <B>CarBoxypeptidaseB> inhiBitor and leech <B>CarBoxypeptidaseB> inhiBitor, Both of which have K values in the nanomolar range. <B>CarBoxypeptidaseB> B has Been isolated from the pancreas of several species including human , cattle , pig , ostrich and others, and human serum and longitudinal muscle layer of cattle small intestine . A staBle enzyme can Be purified from pancreatic acetone powder. Internal proteolysis and degradation may occur if the selected source is pancreatic secretion. The purified enzyme is staBle for years in the precipitated or the frozen states.
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differential scanning calorimetric study of <B>CarBoxypeptidaseB> B pro<B>CarBoxypeptidaseB> B and its gloBular activation domain
FEBS Journal, 1991Co-Authors: Francisco Conejerolara, Josep Vendrell, Pedro L. Mateo, Jose M Sanchezruiz, Francisco J Burgos, Francesc X AvilesAbstract:High-sensitivity differential scanning calorimetry has Been applied to the study of porcine pancreatic <B>CarBoxypeptidaseB> B, the proenzyme and its 81-residue activation domain. The thermal study has Been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has Been found to Be reversiBle and corresponds to that of a typical compact gloBular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversiBle, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two-state irreversiBle model [Sanchez-Ruiz, J.M., Lopez-LacomBa, J.L., Cortijo, M. & Mateo, P.L. (1988) Biochemistry 27, 1648-1652]. Thus the kinetic constants and activation parameters of the denaturation process could Be oBtained and compared to those for other proteins, particularly those of the closely related <B>CarBoxypeptidaseB> A system.
V I Timofeev - One of the best experts on this subject based on the ideXlab platform.
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structure of the complex of <B>CarBoxypeptidaseB> B and n sulfamoyl l arginine
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015Co-Authors: V K Akparov, Nikolay Sokolenko, V I Timofeev, I P KuranovaAbstract:: Porcine pancreatic <B>CarBoxypeptidaseB> B (EC 3.4.23.6) was complexed with a staBle transition-state analogue, N-sulfamoyl-L-arginine, in which an S atom imitates the sp(3)-hyBridized carBon in the scissile-Bond surrogate. Crystals were grown in a form Belonging to the same space group, P41212, as the uncomplexed enzyme. X-ray data were collected to a resolution of 1.25 A. The molecule was refined and the positions of non-H atoms of the inhiBitor and water molecules were defined using difference Fourier maps. The enzyme-inhiBitor complex and 329 water molecules were further refined to a crystallographic R factor of 0.159. The differences in conformation Between the complexed and uncomplexed forms of <B>CarBoxypeptidaseB> B are shown. The inhiBitor is Bound in a curved conformation in the active-site cleft, and the sulfamide group is Bound to the Zn ion in an asymmetric Bidentate fashion. The complex is staBilized By hydrogen Bonds Between the N1/N2 guanidine group of the inhiBitor and the Asp255 carBoxyl of the enzyme. The side-chain CH2 groups of the inhiBitor are in van der Waals contact with Leu203 and Ile247 in the enzyme. This study provides useful clues concerning how the transition state of arginine may Bind to <B>CarBoxypeptidaseB> B and therefore provides an insight into the structural Basis of <B>CarBoxypeptidaseB> B selectivity, which is useful for the rational design of a <B>CarBoxypeptidaseB> with improved selectivity for industrial recomBinant pro-insulin processing.
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Crystallization and preliminary X-ray diffraction study of porcine <B>CarBoxypeptidaseB> B
Crystallography Reports, 2015Co-Authors: V. Kh. Akparov, V I Timofeev, I P KuranovaAbstract:Crystals of porcine pancreatic <B>CarBoxypeptidaseB> B have Been grown in microgravity By the capillary counter-diffusion method through a gel layer. The X-ray diffraction study showed that the crystals Belong to sp. gr. P 4_12_12 and have the following unit-cell parameters: a = B = 79.58 Å, c = 100.51 Å; α = β = γ = 90.00°. The X-ray diffraction data set suitaBle for the determination of the three-dimensional structure at atomic resolution was collected from one of the grown crystals at the SPring 8 synchrotron facility to 0.98 Å resolution.
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preparation crystallization and preliminary x ray diffraction study of mutant <B>CarBoxypeptidaseB> t containing the primary specificity pocket of <B>CarBoxypeptidaseB> B
Crystallography Reports, 2010Co-Authors: Kh V Akparov, V I Timofeev, A M Grishin, I P KuranovaAbstract:RecomBinant G215S, A251G, T257A, D260G, T262D mutant <B>CarBoxypeptidaseB> T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated By X-ray diffraction. RecomBinant mutant <B>CarBoxypeptidaseB> T containing the primary specificity suBsite compositionally identical to that of pancreatic <B>CarBoxypeptidaseB> B crystallizes in the same space group as the natural enzyme. The crystals Belong to sp. gr. P6322; the unit-cell parameters are a = B = 157.867 A, c = 104.304 A, α = β = 90°, γ = 120°. X-ray diffraction data suitaBle for determining the three-dimensional structure at atomic resolution were collected from one crystal.
John Morser - One of the best experts on this subject based on the ideXlab platform.
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plasma <B>CarBoxypeptidaseB> B downregulates inflammatory responses in autoimmune arthritis
Journal of Clinical Investigation, 2011Co-Authors: Jason J Song, Tamsin M Lindstrom, Inyong Hwang, Michael Garcia, Tiffany H Wang, Toshihiko Nishimura, Lei Zhao, John Morser, Michael E Nesheim, Stuart B GoodmanAbstract:The immune and coagulation systems are Both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma <B>CarBoxypeptidaseB> B (CPB), which is activated By the thromBin/thromBomodulin complex, plays a procoagulant role during fiBrin clot formation. However, an antiinflammatory role for CPB is suggested By the recent oBservation that CPB can cleave proinflammatory mediators, such as C5a, Bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerBated inflammatory arthritis in a mouse model of RA, and cleavage of C5a By CPB suppressed the aBility of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at aBrogating the proinflammatory properties of C5a. Additionally, expression of Both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link Between inflammation and coagulation in autoimmune arthritis.
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structures of potent selective peptide mimetics Bound to <B>CarBoxypeptidaseB> B
Acta Crystallographica Section D-biological Crystallography, 2008Co-Authors: Marc Adler, John Morser, Brad O Buckman, Judi Bryant, Zheng Chang, Kieu Chu, Kumar Emayan, Paul Hrvatin, Imadul Islam, Drew SukovichAbstract:This article reports the crystal structures of inhiBitors of the functional form of thromBin-activataBle fiBrinolysis inhiBitor (TAFIa). In vivo experiments indicate that selective inhiBitors of TAFIa would Be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic <B>CarBoxypeptidaseB> B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-Based exopeptidases that are specific for Basic residues. The final development candidate, BX 528, is a potent inhiBitor of TAFIa (2 nM) and has almost no measuraBle effect on the major selectivity target, <B>CarBoxypeptidaseB> N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide Bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural suBstrate. The primary amine in BX 528 forms a salt Bridge to Asp255 at the Base of the S1 pocket. The carBoxylic acid interacts with Arg145 and the sulfonamide is hydrogen Bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 frommore » thiol-Based inhiBitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in Binding affinity. Potency was reclaimed By extending the inhiBitors into the downstream Binding sites for the natural suBstrate.« less