Modulation of the multisubstrate specificity of Thermus maltogenic amylaseby truncation of the N-terminal domain and by a salt-induced shift of the monomer/dimer equilibrium

The relation between the quaternary structure and the substrate specificity of Thermus maltogenic amylase (ThMA) has been investigated. Sedimentation diffusion equilibrium ultracentrifugation and gel filtration analyses, in c ombination with the crystal structure determined recently, have demonstrate d that ThMA existed in a monomer/dimer equilibrium. The truncation of ThMA by removing the N-terminal domain, which is composed of 124 amino acid resi dues, resulted in the complete monomerization of the enzyme (ThMA Delta 124 ) accompanied by a drastic decrease in the activity for beta -cyclodextrin (beta -CD) and a relatively smaller reduction of the activity for starch. D espite the overall low activity of ThMA Delta 124, the activity was higher toward starch than beta -CD, and the ratio of the specific activities towar d these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KCl to wild-type ThMA shifted the monom er/dimer equilibrium toward the monomer. In the presence of 1.0 M KCI, the relative activity of ThMA toward beta -CD decreased to 74%, while that for soluble starch increased to 194% compared to the activities in the absence of KCl. Thus, the ThMA monomer and dimer are both inferred to be enzymatica lly active but with a somewhat different substrate preference. Kinetic para meters of the wild-type and truncated enzymes also are in accordance with t he changes in their specific activities. We thus provide evidence in suppor t of a model, which shows that the relative multisubstrate specificity of T hMA is influenced by the monomer/dimer equilibrium of the enzyme.