Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation

alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from b arley bind with K-i = 0.22 nM. AMY2 is a (beta/alpha)(8)-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-st rand 3 of the (beta/alpha)(8)-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI cr ystal structure [F. Vallee, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg , B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128(AMY2) forms a hydrogen bond with Ser77(BASI), while Asp142(AMY2) makes a salt-bridge with Lys140(BASI). These two enzyme residues are substituted by glutamine and a sparagine, respectively, to assess their contribution in binding of the inh ibitor. These mutations were performed in the well-expressed, inhibitor-sen sitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with K-i = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T12 9R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). T he binding energy was reduced by 2.7-3.0 kcal.mol(-1) as determined from K- i after the mutations R128Q and D142N. This corresponds to loss of a charge d interaction between the protein molecules. In contrast, sensitivity to th e inhibitor was gained (K-i = 7 mu M) by the mutation T129R/K130P in the in sensitive isozyme hybrid. Charge screening raised K-i 14-20-fold for this l atter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effective ly introduced and lost in the different mutant enzyme-inhibitor complexes a nd rational engineering using an inhibitor recognition motif to confer bind ing to the inhibitor mimicking the natural AMY2-BASI complex.