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• W2055352605 abstract "The barley α-amylase/subtilisin inhibitor (BASI) inhibits α-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the α-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on kon, whereas the koff values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the Ki values of some mutants on the AMY2-binding side increased 2–3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca2+ at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca2+ had weaker affinity for AMY2 and reduced sensitivity to the Ca2+ modulation of the affinity. These results suggest that the Ca2+ and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases. The barley α-amylase/subtilisin inhibitor (BASI) inhibits α-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the α-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on kon, whereas the koff values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the Ki values of some mutants on the AMY2-binding side increased 2–3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca2+ at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca2+ had weaker affinity for AMY2 and reduced sensitivity to the Ca2+ modulation of the affinity. These results suggest that the Ca2+ and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases. The double-headed barley α-amylase/subtilisin inhibitor (BASI) 1The abbreviations used are: BASI, barley α-amylase/subtilisin inhibitor; CBD, chitin binding domain; AMY1, barley α-amylase isozyme 1; AMY2, barley α-amylase isozyme 2; RASI, rice α-amylase/subtilisin inhibitor; SPR, surface plasmon resonance; TMA, T. molitor α-amylase; WASI, wheat α-amylase/subtilisin inhibitor; Mes, 4-morpholineethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; PPA, porcine pancreatic α-amylase. of the Kunitz soybean trypsin inhibitor family acts on proteases of the subtilisin family and the endogenous high pI α-amylase (AMY2) but has no effect on the minor isozyme AMY1 that shares 80% sequence identity with AMY2 (1Mundy J. Svendsen I. Hejgaard J. Carlsberg Res. Commun. 1983; 48: 81-91Crossref Scopus (177) Google Scholar, 2Abe J. Sidenius U. Svensson B. Biochem. J. 1993; 293: 151-155Crossref PubMed Scopus (56) Google Scholar, 3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar, 5Nielsen P.K. Bønsager B.C. Fukuda K. Svensson B. Biochim. Biophys. Acta. 2004; 1696: 157-164Crossref PubMed Scopus (53) Google Scholar, 6Svensson B. Fukuda K. Nielsen P.K. Bønsager B.C. Biochim. Biophys. Acta. 2004; 1696: 145-156Crossref PubMed Scopus (146) Google Scholar, 7Rodenburg K. Vallée F. Juge N. Aghajari N. Guo X. Haser R. Svensson B. Eur. J. Biochem. 2000; 267: 1019-1029Crossref PubMed Scopus (42) Google Scholar, 8Nielsen P.K. Bønsager B.C. Berland C.R. Sigurskjold B.W. Svensson B. Biochemistry. 2003; 42: 1478-1487Crossref PubMed Scopus (48) Google Scholar). Under favorable conditions, BASI inhibits the AMY2-catalyzed hydrolysis of starch with a Ki ∼0.1 nm (2Abe J. Sidenius U. Svensson B. Biochem. J. 1993; 293: 151-155Crossref PubMed Scopus (56) Google Scholar, 4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar). This Ki value is in excellent agreement with the KD of 0.07 nm estimated by equilibrium fluorescence titration and stopped-flow kinetics according to a fast 1:1 two-step tight binding reaction (9Sidenius U. Olsen K. Svensson B. Christensen U. FEBS Lett. 1995; 361: 250-254Crossref PubMed Scopus (25) Google Scholar). Surface plasmon resonance (SPR) analysis gave weaker affinities (KD ∼1 nm) presumably due to mass transfer limitations characteristic of the two-phase system and chip surface heterogeneity (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar). In vivo, plausible functions of BASI might be to inhibit AMY2 emerging during premature seed sprouting (1Mundy J. Svendsen I. Hejgaard J. Carlsberg Res. Commun. 1983; 48: 81-91Crossref Scopus (177) Google Scholar, 10D'Ovidio R. Mattei B. Roberti S. Bellincampi D. Biochim. Biophys. Acta. 2004; 1696: 237-244Crossref PubMed Scopus (168) Google Scholar) or the inhibition of proteases from pathogens, e.g. fungi belonging to the genus Fusarium (11Pekkarinen A.I. Jones B.L. J. Agric. Food Chem. 2003; 51: 1710-1717Crossref PubMed Scopus (39) Google Scholar). The in vitro demonstration of a ternary complex of AMY2, BASI, and subtilisin (1Mundy J. Svendsen I. Hejgaard J. Carlsberg Res. Commun. 1983; 48: 81-91Crossref Scopus (177) Google Scholar) is consistent with this latter function. BASI and wheat α-amylase/subtilisin inhibitor (WASI) are highly homologous (92% sequence identity), whereas rice α-amylase/subtilisin inhibitor (RASI), acting on insect α-amylase, shares only 58% sequence identity with BASI (12Mundy J. Heigaard J. Svendsen I. FEBS Lett. 1984; 167: 210-214Crossref Scopus (112) Google Scholar, 13Ohtsubo K-I. Richardson M. FEBS Lett. 1992; 309: 68-72Crossref PubMed Scopus (56) Google Scholar). BASI is assigned to the soybean trypsin inhibitor-like superfamily of β-trefoil fold proteins implicated in various protein-protein interaction roles and shares 20–30% sequence identity with Kunitz-type trypsin inhibitors (6Svensson B. Fukuda K. Nielsen P.K. Bønsager B.C. Biochim. Biophys. Acta. 2004; 1696: 145-156Crossref PubMed Scopus (146) Google Scholar, 14Onesti S. Brick P. Blow D.M. J. Mol. Biol. 1991; 217: 153-176Crossref PubMed Scopus (138) Google Scholar). Six different loops and three β-strands on one side of BASI present residues that interact with both the A and B domains of AMY2 via several hydrogen bonds and salt bridges, resulting in a relatively large protein interface of 2355 Å 2B. Henrissat, afmb.cnrs-mrs.fr/-pedro/CAZY/. (3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) (Fig. 1). Distinct differences in the corresponding AMY1 regions provide a structural rationale for the strict specificity of BASI for AMY2. Most interestingly, a novel feature of the AMY2-BASI complex is the presence of a fully hydrated Ca2+ (Ca503) embedded at the complex interface (3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). This ion seems to mediate binding between inhibitor residues and the catalytic groups in the enzyme via an extended hydrogen bonding network (Figs. 1 and 2B). In addition to Ca503, AMY2 contains three calcium ions bound to domain B (15Kadziola A. Søgaard M. Svensson B. Haser R. J. Mol. Biol. 1998; 278: 205-217Crossref PubMed Scopus (165) Google Scholar, 16Mori H. Bak-Jensen K.S. Svensson B. Eur. J. Biochem. 2002; 269: 5377-5390Crossref PubMed Scopus (30) Google Scholar). In the α-amylase family, or glycoside hydrolase family 13 (17Coutinho P.M. Henrissat B. Recent Advances in Carbohydrate Engineering. The Royal Society of Chemistry, Cambridge, UK1999: 3-14Google Scholar), 2B. Henrissat, afmb.cnrs-mrs.fr/-pedro/CAZY/. the active site is formed between domain B (18Janecek S. Svensson B. Henrissat B. J. Mol. Evol. 1997; 45: 322-331Crossref PubMed Scopus (158) Google Scholar) and the β → α loops from domain A, rendering this part of the protein crucial for substrate binding (15Kadziola A. Søgaard M. Svensson B. Haser R. J. Mol. Biol. 1998; 278: 205-217Crossref PubMed Scopus (165) Google Scholar, 19MacGregor E.A. Janecek S. Svensson B. Biochim. Biophys. Acta. 2001; 1546: 1-20Crossref PubMed Scopus (558) Google Scholar).Fig. 2Stereo view of close-up of two important BASI contact areas (3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). A, Ser77, Tyr131, Lys140, and Asp150 interacting with domain B of AMY2; B, Tyr131, Glu168 and Tyr170 participation in the hydrogen bond network involving water molecules surrounding Ca503 (purple) at the protein interface and the three catalytic acids; C, hydrogen bond network involving Asp156, Arg155, Glu168, Tyr170, and water molecules captured between BASI and AMY2. Ca503 is shown in purple. AMY2 is represented by the molecular surface model in yellow, the catalytic groups in blue, and Pro298 in orange.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The solved structures of complexes between α-amylases and their inhibitors show great structural diversity suggesting important variations in the mode of action of the different inhibitors (for review see Refs. 6Svensson B. Fukuda K. Nielsen P.K. Bønsager B.C. Biochim. Biophys. Acta. 2004; 1696: 145-156Crossref PubMed Scopus (146) Google Scholar and 20Payan F. Leone P. Porciero S. Furniss C. Tahir T. Williamson G. Durand A. Manzanares P. Gilbert H. Juge N. Roussel A. J. Biol. Chem. 2004; 279: 36029-36037Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The majority of these inhibitors, however, elicit inhibition by directly binding to key catalytic groups (21Wiegand G. Epp O. Huber R. Mol. Biol. 1995; 247: 99-110Crossref Scopus (124) Google Scholar, 22Bompard-Gilles C. Rousseau P. Rougé P. Payan F. Structure (Lond.). 1996; 4: 1441-1452Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Strobl S. Maskos K. Wiegand G. Huber R. Gomis-Rüth F.X. Glockshuber R. Structure (Lond.). 1998; 6: 911-921Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) in the target enzyme or by docking on key aromatic groups in proximal subsites (24Desmyter A. Spinelli S. Payan F. Lauwereys M. Wyns L. Muyldermans S. Cambillau C. J. Biol. Chem. 2002; 227: 23645-23650Abstract Full Text Full Text PDF Scopus (145) Google Scholar). In this context, the AMY2-BASI system presents a different example of how inhibition can be attained even though no direct bonds between any of the catalytic groups of the enzyme and the inhibitor are present. Although some binding kinetics and mutagenesis were reported for lectin-like (25Wilcox E.R. Whitaker J.R. Biochemistry. 1984; 23: 1783-1791Crossref PubMed Scopus (46) Google Scholar, 26Mirkov T.E. Evans S.V. Wahlstrom J. Gomez L. Young N.M. Chrispeels M.J. Glycobiology. 1995; 5: 45-50Crossref PubMed Scopus (39) Google Scholar, 27Santimone M. Koukiekolo R. Moreau Y. Le Berre V. Rouge P. Marchis-Mouren G. Desseaux V. Biochim. Biophys. Acta. 2004; 1696: 181-190Crossref PubMed Scopus (52) Google Scholar), cereal-type (28Takase K. Biochemistry. 1994; 33: 7925-7930Crossref PubMed Scopus (23) Google Scholar, 29Alam N. Gourinath S. Dey S. Srinivasan A. Singh T.P. Biochemistry. 2001; 40: 4229-4233Crossref PubMed Scopus (25) Google Scholar), and tendamistat inhibitors (30Vogl T. Brengelmann R. Hinz H.J. Scharf M. Lotzbeyer M. Engels J.W. J. Mol. Biol. 1995; 254: 481-496Crossref PubMed Scopus (55) Google Scholar), thorough analysis is lacking for most systems. An exception is AMY2-BASI, which has been the subject of extensive studies with respect to binding mechanisms and structure/function relationships (2Abe J. Sidenius U. Svensson B. Biochem. J. 1993; 293: 151-155Crossref PubMed Scopus (56) Google Scholar, 5Nielsen P.K. Bønsager B.C. Fukuda K. Svensson B. Biochim. Biophys. Acta. 2004; 1696: 157-164Crossref PubMed Scopus (53) Google Scholar, 6Svensson B. Fukuda K. Nielsen P.K. Bønsager B.C. Biochim. Biophys. Acta. 2004; 1696: 145-156Crossref PubMed Scopus (146) Google Scholar, 9Sidenius U. Olsen K. Svensson B. Christensen U. FEBS Lett. 1995; 361: 250-254Crossref PubMed Scopus (25) Google Scholar). Recently data from mutants of AMY2 residues binding BASI (7Rodenburg K. Vallée F. Juge N. Aghajari N. Guo X. Haser R. Svensson B. Eur. J. Biochem. 2000; 267: 1019-1029Crossref PubMed Scopus (42) Google Scholar), SPR, and isothermal titration calorimetry of AMY2-BASI wild-type protein interactions have demonstrated the effects of ionic strength, pH, and Ca2+ on kinetics and thermodynamics of the complex formation (5Nielsen P.K. Bønsager B.C. Fukuda K. Svensson B. Biochim. Biophys. Acta. 2004; 1696: 157-164Crossref PubMed Scopus (53) Google Scholar, 8Nielsen P.K. Bønsager B.C. Berland C.R. Sigurskjold B.W. Svensson B. Biochemistry. 2003; 42: 1478-1487Crossref PubMed Scopus (48) Google Scholar), but many important questions are still unanswered. The newly established heterologous expression system in Escherichia coli (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar) prompted dissection of the contribution of individual regions and side chains to the overall inhibitory activity of BASI. Another key question addressed is whether the embedded Ca503 and its solvent coordination sphere, as well as other buried solvent molecules, play a significant role in the complex formation and dissociation. This first elaborate mutational analysis of a bifunctional Kunitz-type inhibitor offers insight into the determinants of affinity and the driving forces for complex formation with target enzymes, which paves the way for rational design of other β-trefoil fold family members. Materials—NdeI, BamHI, T4 ligase, and factor Xa were from Promega (Madison, WI); SapI was from New England Biolabs (Beverly, MA), and Expand High Fidelity Polymerase was from Roche Applied Science. Oligonucleotides were from DNA Technologies (Århus, Denmark). Ampicillin, isopropyl β-d-thiogalactopyranoside, dithiothreitol, azoalbumin, and savinase were from Sigma. Insoluble Blue Starch was from Amersham Biosciences. AMY2 (AMY2–2 form; see Refs. 31Svensson B. Mundy J. Gibson R.M. Svendsen I. Carlsberg Res. Commun. 1985; 50: 15-22Crossref Scopus (51) Google Scholar and 32Ajandouz E.H. Abe J. Svensson B. Marchis-Mouren G. Biochim. Biophys. Acta. 1992; 1159: 193-202Crossref PubMed Scopus (77) Google Scholar), BASI (2Abe J. Sidenius U. Svensson B. Biochem. J. 1993; 293: 151-155Crossref PubMed Scopus (56) Google Scholar), and TMA (23Strobl S. Maskos K. Wiegand G. Huber R. Gomis-Rüth F.X. Glockshuber R. Structure (Lond.). 1998; 6: 911-921Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) were purified from kilned malt (cv. Menuet), mature barley seeds (cv. Piggy), and Tenebrio molitor larvae (gift from S. O. Andersen), respectively. E. coli DH5α and BL21 (DE3) were from Stratagene (Amsterdam, Netherlands). pCR 2.1-TOPO vector, TOPO cloning kit (including competent cells), Pichia pastoris GS115, and pHIL-D2 shuttle vector were from Invitrogen. pET11a was from Novagen (Madison, WI), and the IMPACT intein cloning kit including pTYB1 vector was from New England Biolabs. pUC19 containing BASI cDNA from cv. Piggy (33Leah R. Mundy J. Plant Mol. Biol. 1989; 12: 673-682Crossref PubMed Scopus (88) Google Scholar) (GenBank™ accession number Z12961) was used as template for PCR. Generation of Mutants—S77A, K140L/K140N, E168Q/E168T, and Y170F/Y170P BASI with C-terminal intein-CBD tag (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar, 34Chong S. Mersha F.B. Comb D.G. Scott M.E. Landry D. Vence L.M. Perler F.B. Benner J. Kucera R.B. Hirvonen C.A. Pelletier J.J. Paulus H. Xu M.-Q. Gene (Amst.). 1997; 192: 277-281Crossref Scopus (510) Google Scholar), and Y131F, D150N, D156K, E168Q/Y170P, Y87A, T89A, S93A, and E95Q with N-terminal His6 tags were produced in E. coli (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar, 35Hochuli E. Bannewarth W. Doebli H. Gentz R. Stueber D. J. Chromatogr. 1988; 411: 177-184Crossref Scopus (980) Google Scholar). The following internal mutagenic primers were used to incorporate mutants (underlined): S77A, 5′-CGGACGTCGGTCGCCAGCCGGATGA-3′; Y131F, 5′-CGGCGCCGCTAAACTTCTCGATGCGG-3; K140L, 5′-CACGACATCAGCAGGTACTCGTGC-3′; K140N, 5′-CGCACGACATCAGGTTGTACTCGTGC-3′; D150N, 5′-CACGCCGAGGTTCTGGCACCAGTCC-3′; Y87A, 5′-GCAGACACGTCGTGGCGGCGCGGAAGG-3′; T89A, 5′-GGACTGCAGACACGCCGTGTAGGCGCGG-3′; S93A, 5′-GCCACTCAGTGGCCTGCAGACACG-3′; E95Q, 5′-GTCGATGTGCCACTGAGTGGACTGCAG-3′; and D156K, 5′-CCCTTGAGTTTCCTGAACAGC. For the 5′-primers encoded mutations at positions 168 and 170: E168Q, 5′-GCTCTTCCGCAAGCGGGCGGCGCCTTCTTGAACACGACGACATGGTATGGCTGGGTGG-3′; E168T, 5′-GCTCTTCCGCAAGCGGGCGGCGCCTTCTTGAACACGACGACATGGTATGGGTTGGTGG-3′; Y170F, 5′-GCTCTTCCGCAAGCGGGCGGCGCCTTCTTGAACACGACGACATGAAATGGC-3′; Y170P, 5′-GCTCTTCCGCAAGCGGGCGGCGCCTTCTTGAACACGACGACATGCGGTGGC-3′; and E168Q/Y170P, 5′-CATATGCCGCAAGCGGGCGGCGCCTTCTTGAACACGACGACATGCGGTGGCTGGGTG-3′ (SapI and NdeI sites in boldface). Mutations were generated using standard PCR methods and the cDNA was cloned into the pTYB1 vector (intein-system) or the pET11a vector. E. coli BL21 was transformed with the modified vectors and selected on ampicillin (100 μg × ml–1) LB plates. Cells grown at 20 °C (0.5 liters) were induced by 4 μm isopropyl β-d-thiogalactopyranoside at A600 = 0.6, harvested after 24 h by centrifugation, resuspended in 10 ml of intein-CBD tag buffer (20 mm Hepes, pH 8.0, 0.5 m NaCl, 1 mm EDTA) and His6 tag buffer (30 mm Tris, pH 8.0, 10 mm imidazole, 0.5 m NaCl), and sonicated. Intein-CBD tag BASI was applied to a chitin column (2 ml), left in 30 mm dithiothreitol overnight to autohydrolyze (34Chong S. Mersha F.B. Comb D.G. Scott M.E. Landry D. Vence L.M. Perler F.B. Benner J. Kucera R.B. Hirvonen C.A. Pelletier J.J. Paulus H. Xu M.-Q. Gene (Amst.). 1997; 192: 277-281Crossref Scopus (510) Google Scholar), eluted by 20 mm Hepes, pH 8.0, 0.05 m NaCl, 1 mm EDTA, and purified on Superose 12 HP 10/30. His6 tag BASI was purified on a HiTrap HP nickel column (1 ml; Amersham Biosciences), eluted by a 30–500 mm imidazole gradient using an ÄKTAexplorer chromatograph (Amersham Biosciences), and finally purified on Sephacryl S-100 HR (Amersham Biosciences; 60 × 1.6 cm). Optional factor Xa cleavage was performed as described (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar). Protein Characterization—SDS-PAGE (4–12%) and isoelectric focusing, pH 3–10 (NOVEX and Invitrogen), were performed as recommended by the manufacturers. Protein concentrations were calculated from amino acid contents of hydrolysates (0.2–1.0 nmol; 6 m HCl, 110 °C, 22 h) determined on a Biochrom 20 analyzer (Amersham Biosciences). Circular dichroism was measured with a Jasco J-810 spectropolarimeter dichrograph at 25 °C. Ten spectra were scanned at 250 to 190 nm by using a quartz cell (path length 1.0 mm) and scaled in the protein concentration range 1.5–5 μm. AMY2 Inhibition—BASI mutants or wild types (12 concentrations in the range 0.3 nm to 3 μm were chosen to match the inhibitory activity) were preincubated with AMY2 (3.2 nm; 400 μl) for 30 min at 37 °C in either 40 mm Tris-HCl, pH 8.0, 5 mm CaCl2, 0.05% bovine serum albumin (optimal for inhibition) or 20 mm Hepes-NaOH, pH 6.8, 5 mm CaCl2, 0.2 m NaCl (suboptimal). The preincubation mixture (200 μl) was added to insoluble Blue Starch (6.25 mg in the corresponding buffer; 800 μl) and incubated at 37 °C. After 30 min, the reaction was stopped by 0.5 m NaOH (250 μl) and centrifuged, and the absorbance was measured at 620 nm (MRX-TC Revelation microtiter plate reader, Dynex Technologies) (2Abe J. Sidenius U. Svensson B. Biochem. J. 1993; 293: 151-155Crossref PubMed Scopus (56) Google Scholar). The Km values of AMY2 were 1.0 × 10–3 and 1.3 × 10–3 g × ml–1 under optimal and suboptimal conditions, respectively, and 1.6 × 10–3, 1.1 × 10–3, and 2.5 × 10–3 g × ml–1 at ∼1 μm Ca2+ (present in the buffer without CaCl2 addition), 1 mm, and 20 mm CaCl2 (pH 8.0), as determined by fitting rates at seven insoluble Blue Starch concentrations (0.2–5 mg × ml–1) to the Michaelis-Menten equation. Test for Inhibition of Yellow Meal Worm (T. molitor) α-Amylase—Up to 42 μm E168Q, Y170P, E168Q/Y170P, or wild-type BASI and TMA (21 nm) were preincubated at 37 °C (30 min) in 50 mm sodium acetate, pH 5.5, 5 mm CaCl2, 0.1 m NaCl, 0.05% bovine serum albumin (400 μl); and insoluble Blue Starch was added (6.25 mg in 600 μl). The reaction was stopped after 30 min and activity quantified as for AMY2. Inhibition of Savinase—BASI Y87A, T89A, S93A, E95Q, or wild type (10 concentrations in the range 60 nm to 2 μm) were preincubated with savinase (125 nm; 200 μl) for 20 min at 37 °C in 50 mm Bicine, pH 8.5, 2 mm CaCl2, 0.1 m KCl, and added azoalbumin (15 mg × ml–1; 200 μl) (13Ohtsubo K-I. Richardson M. FEBS Lett. 1992; 309: 68-72Crossref PubMed Scopus (56) Google Scholar). The reaction was stopped after 45 min by 5% trichloroacetic acid (600 μl), left 30 min, and centrifuged. The supernatant was mixed 1:1 with 10% NaOH, and A492 was measured as above. The Km of savinase for azoalbumin was 14 mg × ml–1 as determined from the initial hydrolysis rates at seven substrate concentrations (0.5–12.5 mg × ml–1). Calculation of Ki—Determination of the intrinsic Ki = Ki,app × (1 + S/Km)–1 (36.Bieth, J. (1974) in Bayer-Symposium V “Protein Inhibitors” (Fritz, M., Tschesche, M., Greene, L J., and Truscheit, E., eds) pp. 463–469, Springer-Verlag, BerlinGoogle Scholar) assumed equilibrium between AMY2, BASI, substrate (Blue Starch), and the AMY2-BASI complex. Inhibition curves showed % inhibition, 100 × (1 –(Acti/Act0)) versus BASI:AMY2 (molar ratio); Act0 and Acti are activities in the absence and presence of BASI. Ki,app was obtained from the slope of 1/Acti versus [BASI0]/(1–Acti), using the intercept on the y axis indicated the enzyme concentration (according to Fig. 5 in Ref. 36.Bieth, J. (1974) in Bayer-Symposium V “Protein Inhibitors” (Fritz, M., Tschesche, M., Greene, L J., and Truscheit, E., eds) pp. 463–469, Springer-Verlag, BerlinGoogle Scholar). The Ki value for inhibition of savinase was determined by using the above equations with the appropriate Km values and substrate (azoalbumin). Surface Plasmon Resonance—Binding kinetics of BASI and either AMY2 or savinase was determined by using a BIAcore 3000 (Biosensor, Amersham Biosciences) (4Bønsager B.C. Prætorius-Ibba M. Nielsen P.K. Svensson B. Protein Expression Purif. 2003; 30: 185-193Crossref PubMed Scopus (18) Google Scholar, 8Nielsen P.K. Bønsager B.C. Berland C.R. Sigurskjold B.W. Svensson B. Biochemistry. 2003; 42: 1478-1487Crossref PubMed Scopus (48) Google Scholar). The sensor chips were charged by 300–1000 response units of either biotinylated AMY2 (streptavidin-sensor chips), BASI, or savinase bound by using the amine coupling procedure (CM5 sensor chips) (8Nielsen P.K. Bønsager B.C. Berland C.R. Sigurskjold B.W. Svensson B. Biochemistry. 2003; 42: 1478-1487Crossref PubMed Scopus (48) Google Scholar). Sensorgrams (response units versus time) in duplicate were recorded at a flow rate of 30 μl × min–1 at 25 °C, using five analyte concentrations (20–300 nm) in 10 mm Hepes, pH 8.0, 5 mm CaCl2, and 0.005% surfactant P20. The effect of Ca2+ was measured in 10 mm Mes, pH 6.5, 0.005% surfactant P20. The association and dissociation were monitored for 4 and 15 min, respectively, and the chip was regenerated by 10 mm sodium acetate, pH 5.0 (AMY2), or by 5 min of prolonged dissociation (savinase). Sensorgrams were analyzed using BIAevaluation version 3.1 software applying a single site 1:1 (Langmuir) binding model: A + B ⇄ AB) and deriving koff, kon, and KD values (8Nielsen P.K. Bønsager B.C. Berland C.R. Sigurskjold B.W. Svensson B. Biochemistry. 2003; 42: 1478-1487Crossref PubMed Scopus (48) Google Scholar). Binding energy differences were calculated using ΔΔG = –RT ln(KD,mut/KD,wt) (37Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (256) Google Scholar). Illustrations—Molecular graphics were made using AMY2-BASI accession 1AVA (3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) in the Protein Data Bank (38Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar), Swiss-PdbViewer (39Kaplan W. Littlejohn T.G. Brief Bioinform. 2001; 2: 195-197Crossref PubMed Scopus (335) Google Scholar) (us.expasy.org/spdbv/), and POV-Ray (www.povray.org/). Multiple sequence alignments were made using the GenBank™ entries and the ClustalW program at the European Bioinformatics Institute (www.ebi.ac.uk/clustalw/). Choice and Production of BASI Mutants—The structure of AMY2-BASI (3Vallée F. Kadziola A. Bourne Y. Juy M. Rodenburg K. Svensson B. Haser R. Structure (Lond.). 1998; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), sequence alignment of BASI, WASI, and RASI (Fig. 3), and the structure of proteinase K/WASI (40Pal G.P. Kavounis C.A. Jany K.D. Tsernoglou D. FEBS Lett. 1994; 341: 167-170Crossref PubMed Scopus (26) Google Scholar) guided mutations in four different regions of BASI (Figs. 1 and 2). Three of the four regions were located at the AMY2-BASI interface, and the fourth was on the opposite side of the inhibitor, which is suggested to be involved in protease inhibition. In the case of the AMY2-BASI interface, mutations were focused on two regions of BASI, the first of which is in contact with AMY2 domain B, and the other overlooks the active site of the enzyme (Fig. 2, A and B). In addition, one charge-reversed mutation was introduced in a third region of BASI in contact with AMY2 domain A but fairly distant from the active site (Figs. 1 and 2C). In the first region of BASI, Ser77, Tyr131, Lys140, and Asp150, which form various hydrogen bonds to the two previously mutated AMY2 residues Arg128 and Asp142 (Table I; Figs. 2, A and B and 3) and also to Gly144, were replaced mostly by sterically similar side chains (41Bordo D. Argos P. J. Mol. Biol. 1991; 217: 721-729Crossref PubMed Scopus (336) Google Scholar). Glu168 and Tyr170 were the targets of mutations in the second binding region located at the center of the binding interface. These residues are involved in an extended hydrogen bonding network comprising the hydration shell of the embedded Ca503 and the three AMY2 catalytic groups Asp179, Glu204, and Asp289 (Table I; Fig. 2B). The only AMY2-BASI direct bond involving these residues is between Glu168 in BASI and Lys182 in AMY2 that in turn forms a hydrogen bond to a substrate glucosyl residue at subsite +2 (15Kadziola A. Søgaard M. Svensson B. Haser R. J. Mol. Biol. 1998; 278: 205-217Crossref PubMed Scopus (165) Google Scholar, 19MacGregor E.A. Janecek S. Svensson B. Biochim. Biophys. Acta. 2001; 1546: 1-20Crossref PubMed Scopus (558) Google Scholar). Moreover, Glu168 and Tyr170 correspond to glutamine and proline in the rice homologue RASI (Fig. 3) that inhibits insect α-amylase (13Ohtsubo K-I. Richardson M. FEBS Lett. 1992; 309: 68-72Crossref PubMed Scopus (56) Google Scholar), and the RASI mimics E168Q, Y170P, and E168Q/Y170P were designed to assess if they could confer inhibitory activity on insect α-amylase. In the third region Asp156 (Fig. 2C), located at the periphery of the complex and having only a water-mediated bond to AMY2, was charge-reversed to probe how changes in charge density in that region would affect the binding" @default.
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• W2055352605 title "Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor" @default.
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