Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor*

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.

[1]  A. Minton,et al.  Analysis of mass transport-limited binding kinetics in evanescent wave biosensors. , 1996, Analytical biochemistry.

[2]  Š. Janeček,et al.  Relationship of sequence and structure to specificity in the α-amylase family of enzymes , 2001 .

[3]  K. Takase.  Site-directed mutagenesis reveals critical importance of the catalytic site in the binding of alpha-amylase by wheat proteinaceous inhibitor. , 1994, Biochemistry.

[4]  K. Ohtsubo,et al.  The amino acid sequence of a 20 kDa bifunctional subtilisin/α‐amylase inhibitor from brain of rice (Oryza saliva L.) seeds , 1992 .

[5]  B. Svensson,et al.  Partial amino acid sequences of α-amylase isozymes from barley malt , 1985 .

[6]  P. Argos,et al.  Suggestions for "safe" residue substitutions in site-directed mutagenesis. , 1991, Journal of molecular biology.

[7]  W. Delano.  Unraveling hot spots in binding interfaces: progress and challenges. , 2002, Current opinion in structural biology.

[8]  B. Svensson,et al.  Purification and characterization of the β-trefoil fold protein barley α-amylase/subtilisin inhibitor overexpressed in Escherichia coli , 2003 .

[9]  J. Mundy,et al.  Characterization of a bifunctional wheat inhibitor of endogenous α‐amylase and subtilisin , 1984 .

[10]  B. Svensson,et al.  The structure of barley alpha-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs. , 2003, Structure.

[11]  J. Whitaker,et al.  Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase. , 1984, Biochemistry.

[12]  Walter F Stafford,et al.  Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation. , 2004, Journal of molecular biology.

[13]  T. Vogl,et al.  Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat. , 1995, Journal of molecular biology.

[14]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[15]  Birte Svensson,et al.  Barley α-amylase/subtilisin inhibitor: structure, biophysics and protein engineering , 2004 .

[16]  E. Hochuli,et al.  New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. , 1987, Journal of chromatography.

[17]  B. Jones,et al.  Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum. , 2003, Journal of agricultural and food chemistry.

[18]  Mengsu Yang,et al.  Effect of hydrogel matrix on binding kinetics of protein–protein interactions on sensor surface , 2002 .

[19]  R. D'Ovidio,et al.  Polygalacturonases, polygalacturonase-inhibiting proteins and pectic oligomers in plant-pathogen interactions. , 2004, Biochimica et biophysica acta.

[20]  J. Bieth.  Some Kinetic Consequences of the Tight Binding of Protein-Proteinase-Inhibitors to Proteolytic Enzymes and their Application to the Determination of Dissociation Constants , 1974 .

[21]  M. Sanderson.  High-speed digital microscopy. , 2000, Methods.

[22]  B. Svensson,et al.  Proteinaceous α-amylase inhibitors , 2004 .

[23]  B. Svensson,et al.  Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates. , 1992, Biochimica et biophysica acta.

[24]  D. Rigden,et al.  Plant α‐amylase inhibitors and their interaction with insect α‐amylases , 2002 .

[25]  B. Svensson,et al.  Arginine is essential for the α-amylase inhibitory activity of the α-amylase/subtilisin inhibitor (BASI) from barley seeds , 1993 .

[26]  J. Mundy,et al.  Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization , 1983 .

[27]  Alain Roussel,et al.  The Dual Nature of the Wheat Xylanase Protein Inhibitor XIP-I , 2004, Journal of Biological Chemistry.

[28]  N. Juge,et al.  Specific inhibition of barley α‐amylase 2 by barley α‐amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation , 2000 .

[29]  T. Littlejohn,et al.  Swiss-PDB Viewer (Deep View). , 2001, Briefings in bioinformatics.

[30]  C. Bompard-Gilles,et al.  Substrate mimicry in the active center of a mammalian alpha-amylase: structural analysis of an enzyme-inhibitor complex. , 1996, Structure.

[31]  P. Schuck,et al.  Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. , 1997, Annual review of biophysics and biomolecular structure.

[32]  R. Huber,et al.  The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat. , 1995, Journal of molecular biology.

[33]  P. Rougé,et al.  Porcine pancreatic α-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (α-AI1) and structural changes in the α-amylase inhibitor complex , 2004 .

[34]  B. Svensson,et al.  Barley α-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the β→α loop 2 of the catalytic (β/α)8-barrel and is critical for activity and substrate specificity , 2002 .

[35]  David G Myszka,et al.  Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. , 2004, Analytical biochemistry.

[36]  B. Henrissat,et al.  Domain Evolution in the α-Amylase Family , 1997, Journal of Molecular Evolution.

[37]  A. Fersht,et al.  Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. , 1983, Biochemistry.

[38]  B. Svensson,et al.  Molecular structure of a barley alpha-amylase-inhibitor complex: implications for starch binding and catalysis. , 1998, Journal of molecular biology.

[39]  D Tsernoglou,et al.  The three‐dimensional structure of the complex of proteinase K with its naturally occurring protein inhibitor, PKI3 , 1994, FEBS letters.

[40]  Birte Svensson,et al.  Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry. , 2003, Biochemistry.

[41]  T. Mirkov,et al.  Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad. , 1995, Glycobiology.

[42]  R. Weselake,et al.  Effect of endogenous barley α-amylase inhibitor on hydrolysis of starch under various conditions , 1985 .

[43]  A. Bogan,et al.  Anatomy of hot spots in protein interfaces. , 1998, Journal of molecular biology.

[44]  D G Myszka,et al.  Advances in surface plasmon resonance biosensor analysis. , 2000, Current opinion in biotechnology.

[45]  Robert Huber,et al.  A novel strategy for inhibition of α-amylases: yellow meal worm α-amylase in complex with the Ragi bifunctional inhibitor at 2.5 å resolution , 1998 .

[46]  T. Singh,et al.  Substrate−Inhibitor Interactions in the Kinetics of α-Amylase Inhibition by Ragi α-amylase/Trypsin Inhibitor (RATI) and Its Various N-Terminal Fragments† , 2001 .

[47]  B. Svensson,et al.  Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution. , 1998, Structure.

[48]  R. Leah,et al.  The bifunctional α-amylase/subtilisin inhibitor of barley: nucleotide sequence and patterns of seed-specific expression , 1989, Plant Molecular Biology.

[49]  Yili Li,et al.  Dissection of binding interactions in the complex between the anti-lysozyme antibody HyHEL-63 and its antigen. , 2003 .

[50]  B. Svensson,et al.  Stopped‐flow kinetic studies of the reaction of barley α‐amylase/subtilisin inhibitor and the high pI barley α‐amylase , 1995 .

[51]  P. Brick,et al.  Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds. , 1991, Journal of molecular biology.