A single amino acid substitution that determines the IEF band pattern of barley ß-amylase

Barley beta-amylase exhibits two distinct band patterns after isoelectric focusing (IEF), termed Sd1 and Sd2. A comparison of the deduced amino acid sequences revealed five amino acid differences between the two types of beta-amylase. To investigate whether the two band patterns are due to these amino acid substitutions, four Sd2 mutants (Sd2-R115C, Sd2-D165E, Sd2-L347S and Sd2-V430A) were constructed by site-directed mutagenesis. The analysis of IEF band patterns of mutant and wild-type beta-amylases revealed that only the replacement of R115 with cysteine converted the Sd2 band pattern to the Sd1 one. The contribution of the R115C substitution to the IEF band pattern of barley beta-amylase was further confirmed by generating a Sd1 mutant (Sd1-C115R), where cysteine at this position was in turn replaced by arginine. As a result of this mutation, the Sd1 band pattern was converted into the Sd2 pattern. Calculation of the electrostatic potential and reducing agent treatment revealed that the R115C substitution altered both the net charge on the protein surface and intermolecular interactions by disulfide bonds, thereby altering the IEF band pattern of barley beta-amylase © 2002 Elsevier Science Ltd.