Structural studies on aldolase isozymes through protein engineering.
Enzymatic studies on aldolase isozymes have been carried out by techniques of protein engineering. Site-directed mutagenesis helps us to verify the roles of amino acid residues in catalytic reactions. Chimeric fusion proteins give us information about the regions which specify the characteristics of the isozymes. The results are: (1) In aldolase A, COOH terminal Tyr and Lys-107 residues play important roles in catalysis, especially in binding of FDP. (2) Aspartic acid at the 128th residue in aldolase A is essential to thermostability; no other residue such as glutamic acid can substitute for it. (3) Studies on chimeric fusion proteins indicate that the C-terminal region (including C-terminus Tyr) or aldolase A is responsible for its substrate specificity, which is not seen in aldolase B. (4) A region near NH2 terminus in aldolase B determines its specific structure. (5) The region including His-107, Asp-128, and Tyr-137 (B-A junction of BA137) is located in a turn which is exposed outward (a model architecture by Sygusch et al [1987]). In BA137, this region would be constrained, and play a significant role in catalysis, thermostability, etc. (6) Tertiary structure of aldolase B seems to be dissimilar to that of aldolase A.
Department of Biochemistry, Saga Medical School, Japan.
Y
Y
Takasaki
Y
Y
Kitajima
I
I
Takahashi
M
M
Sakakibara
T
T
Mukai
K
K
Hori
D000595:Amino Acid Sequence; D000743:Anemia, Hemolytic / Q000201:enzymology; D000818:Animals; D002678:Chimera; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D005634:Fructose-Bisphosphate Aldolase