Human aldolase A of a hemolytic anemia patient with Asp-128----Gly substitution: characteristics of an enzyme generated in E. coli transfected with the expression plasmid pHAAD128G.
Aldolase A derived from a hemolytic anemia patient with aldolase A deficiency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme [Kishi et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8623-8627]. 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 analysis 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 [aldolase A(D-E)] and Ser for Asp [aldolase A(D-S)], respectively, at the position were constructed by site-directed mutagenesis and characterized. These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.
Takasaki
Y
Y
Department of Biochemistry, Saga Medical School.
Takahashi
I
I
Mukai
T
T
Hori
K
K
D000595:Amino Acid Sequence; D000743:Anemia, Hemolytic / Q000201:enzymology*; D001224:Aspartic Acid / Q000032:analysis; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000172:deficiency / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010957:Plasmids; D011489:Protein Denaturation; D014162:Transfection; D014357:Trypsin
D007527:Isoenzymes; D001224:Aspartic Acid; D014357:Trypsin; D005634:Fructose-Bisphosphate Aldolase; D005998:Glycine