Expression, purification, and characterization of natural mutants of human aldolase B. Role of quaternary structure in catalysis.
Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.
Rellos
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Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom.
Sygusch
J
J
Cox
T M
TM
D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase