Abstract: Characteristics of two rice alpha-amylases Amy1A and Amy3D , and those of two chimeric enzymes Amy1A/3D and Amy3D/1A , engineered from the two isozymes , were compared in the light of the functional roles of protein domains in alpha-amylase .
The enzymes that have an Amy1A-type N-terminal domain , Amy1A and Amy1A/3D , showed high activity against soluble starch , while the enzymes that have an Amy3D-type barrel structure , Amy3D and Amy1A/3D , showed high activity in oligosaccharide hydrolysis .
Rigidity of protein folding also significantly affected the enzyme activity in both soluble starch and oligosaccharide hydrolysis .
Thus , the present work suggests that the structure of the N-terminal domain is important for stability and soluble starch hydrolysis , while the barrel structure that forms the active site significantly affects enzyme activities in oligosaccharide degradation .
We have already characterized two rice alpha-amylase isozymes , Amy1A and Amy3D , and a chimeric enzyme engineered from these two isozymes , Amy1A/3D ( Terashima et al 1995 , 1996a , b ) .
In spite of the high homology ( 70% ) of their amino acid sequences , Amy1A and Amy3D showed distinct differences in their enzymatic characteristics .
The chimeric enzyme Amy1A/3D , which consists of an Amy1A-type N-terminal domain and an Amy3D-type barrel structure , inherited enzymatic characteristics from the both isozymes .
In this work , one other chimeric enzyme , Amy3D/1A , which is the counterpart of Amy1A/3D , has been characterized .
The characteristics of these four enzymes are discussed in the light of the functional roles of protein domains . |