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J. Biochem, 1990, Vol. 108, No. 2 153-157
© 1990 Japanese Biochemical Society

research-article

Human Aldolase A of a Hemolytic Anemia Patient with Asp-128->Substitution: Characteristics of an Enzyme Generated in E. coli Transfected with the Expression Plasmid pHAAD128G1

Yozo Takasaki*,2, Isamu Takahashi*, Tsunehiro Mukal** and Katsuji Hori*

*Department of Biochemistry, Saga Medical School Saga, Saga 840-01
**Department of Bioscience, National Cardiovascular Center Institute Suita, Osaka 565

2To whom correspondence should be addressed

Aldolase A derived from a hemolytic anemia patient with aldolase A decificency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme [Kishi et at. (1987) Proc. Nati. Acad. Sci. U.S.A. 84, 8823–8627]. We constructed an Escherichia coli expression plasmid, pHAAD128G, which carries the mutant aldolase A [aldolase A(D-G)] cDNA, and the enzyme generated in E. coli transfected with the expression plasmid was purified and characterized. Conversion of Asp to Gly at the 128th position in the enzyme rendered the enzyme thermolabile and susceptible to tryptic digestion. CD spectra analyses also revealed that the mutant enzyme had a remarkable conformation change with a decrease of regular form In the molecule. Addition of glycerol or some other polyalcohols during thermal treatment protected this altered enzyme (but not the normal enzyme) against denaturation and activity decrease. In order to determine the function of the amino acid residue at the 128th position, two artificial mutant enzymes with the substitutions of Glu for Asp [ A(D-E)] and Ser for Asp [ A(D-S)], respectively, at the position were constructed by site-directed mutagenesis and character ized. These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. This work was also supported by a grant from the Uehara Memorial Foundation.


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