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.
The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.
Nielsen
Peter K
PK
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Bønsager
Birgit C
BC
Berland
Carolyn R
CR
Sigurskjold
Bent W
BW
Svensson
Birte
B
D002118:Calcium / Q000737:chemistry; D002151:Calorimetry / Q000379:methods; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D001467:Hordeum / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors*; D020349:Surface Plasmon Resonance / Q000379:methods; D013696:Temperature; D013816:Thermodynamics; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry* / Q000378:metabolism; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D010940:Plant Proteins; C092772:barley alpha-amylase-subtilisin inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D000516:alpha-Amylases; D012697:Serine Endopeptidases; D013381:Subtilisins; C032923:microbial serine proteinases; D002118:Calcium