Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B.
In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)
Takahashi
I
I
Department of Biochemistry, Saga Medical School.
Takasaki
Y
Y
Hori
K
K
D000595:Amino Acid Sequence; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D013696:Temperature
D007527:Isoenzymes; D005634:Fructose-Bisphosphate Aldolase; D003545:Cysteine