Modulation of the multisubstrate specificity of Thermus maltogenic amylase by 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 combination with the crystal structure determined recently, have demonstrated 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 residues, resulted in the complete monomerization of the enzyme (ThMADelta124) accompanied by a drastic decrease in the activity for beta-cyclodextrin (beta-CD) and a relatively smaller reduction of the activity for starch. Despite the overall low activity of ThMADelta124, the activity was higher toward starch than beta-CD, and the ratio of the specific activities toward these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KCl to wild-type ThMA shifted the monomer/dimer equilibrium toward the monomer. In the presence of 1.0 M KCl, 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 enzymatically active but with a somewhat different substrate preference. Kinetic parameters of the wild-type and truncated enzymes also are in accordance with the changes in their specific activities. We thus provide evidence in support of a model, which shows that the relative multisubstrate specificity of ThMA is influenced by the monomer/dimer equilibrium of the enzyme.