ARGININE IS ESSENTIAL FOR THE ALPHA-AMYLASE INHIBITORY ACTIVITY OF THE ALPHA-AMYLASE SUBTILISIN INHIBITOR (BASI) FROM BARLEY-SEEDS/

Treatment of barley alpha-amylase/subtilisin inhibitor (BASI) with rea gents specific for arginine, histidine, methionine and tyrosine residu es and amino and carboxyl groups indicates that an arginine residue(s) is essential for its action on the target enzyme barley alpha-amylase 2. Phenylglyoxal modified eight out of 12 arginine residues in BASI. Kinetic analysis shows that the inactivation of BASI follows a pseudo- first-order reaction and is due to reaction with one molecule of pheny lglyoxal; the second-order rate constant is determined to be 2.95 M-1 . min-1. At pH 8.0, BASI and barley alpha-amylase 2 form an inactive 1 :1 complex. The K(i) value of this association is 2.2 x 10(-10) M. The alpha-amylase protects four arginine residues and also the alpha-amyl ase inhibitory activity of BASI against phenylglyoxal. When BASI from the phenylglyoxal-modified target enzyme-inhibitor complex is isolated and subjected to a second treatment with phenylglyoxal, four addition al arginine residues are modified, with concomitant loss of the inhibi tory activity. These results are discussed in relation to a three-dime nsional model of BASI based on the known structure of the correspondin g inhibitor from wheat.