title: Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli.
abstract: To study the structure/function relationship and enzymatic properties of human aldolase C, we have constructed an Escherichia coli expression plasmid, pHAC11, for the isozyme. E. coli cells carrying this plasmid produced enzymatically active human aldolase C. The kcat and Km values for fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P) of the recombinant enzyme were found to be similar to those of authentic aldolase C from human brain. The Fru-1,6-P2/Fru-1-P activity ratio of the recombinant enzyme is approximately 13.5, which is comparable to that of the recombinant rat aldolase C, but is slightly higher than those of rat brain and hepatoma aldolases C. The substitution of Ser for the carboxyl-terminal Tyr (Tyr-363) of the recombinant enzyme caused a marked decrease in that of Fru-1,6-P2, with little change in that of Fru-1-P. The activity ratio changed from 13.5 for the normal enzyme to 3.8 for the engineered enzyme. Human aldolase C was found to form tetrameric hybrids with aldolase B in vivo when these enzymes were coexpressed in E. coli cells.
authors.affiliation: Department of Biochemistry, Saga Medical School.
authors.forename: T
authors.initials: T
authors.lastname: Kusakabe
authors.forename: K
authors.initials: K
authors.lastname: Motoki
authors.forename: K
authors.initials: K
authors.lastname: Hori
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli; D005634:Fructose-Bisphosphate Aldolase / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013329:Structure-Activity Relationship
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase