Expression, purification, and characterization of natural mutants of humanaldolase B - Role of quaternary structure in catalysis

Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyz e the reversible cleavage of phosphofructose esters into cognate triose (ph osphates). Three vertebrate isozymes of Class I aldolase have arisen by gen e duplication and display distinct activity profiles with fructose 1,6-bisp hosphate and with fructose l-phosphate, We describe the biochemical and bio physical characterization of seven natural human aldolase B variants, ident ified in patients suffering from hereditary fructose intolerance and expres sed as recombinant proteins in E, call, from which they were purified to ho mogeneity. The mutant aldolases were all missense variants and could be cla ssified into two principal groups: catalytic mutants, with retained tetrame ric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into sub units with greatly impaired enzymatic activity (A149P, A174D, L256P, and N3 34K), Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for mainta ining its full catalytic function.