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)
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Takahashi et al. (1989) studied this question.