Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of alpha-1,4 glucan. From our previous study [Fujii et A, Appl. Environ. Microbiol., 71, 5823-5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 angstrom away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.