Differential usage of the carboxyl-terminal region among aldolase isozymes.
Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases. Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349. Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm. Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency. Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates. Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequence insertion of residues 346-349 modifies substrate and aldehyde interaction with C6 phosphate binding locus. Low intrinsic susceptibility to carboxypeptidase A degradation of the COOH terminus in liver aldolase is consistent with tight association of this COOH terminus in a conformation unfavorable for promoting high catalytic activity. Efficient carbanion protonation promoted by specific sequences 357-362 represents a mechanistic feature which distinguishes catalytically active maize and muscle isozymes from less active liver isozyme. Conservation of active site residues among aldolases suggests that isozyme diversity among aldolases arose from divergent evolution of the COOH-terminal sequence.
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
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L
Berthiaume
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DR
Tolan
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J
Sygusch
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004330:Drosophila / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005634:Fructose-Bisphosphate Aldolase