Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala(337) -> Val) in fructose intolerance

A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala(337) --> Val in homozygous form. This mutation was also detected independently in two othe r affected individuals who were compound heterozygotes for the prevalent al dolase B allele, Ala(149) --> Pro, indicating that the mutation causes aldo lase B deficiency. To test for the effect of the mutation, catalytically ac tive wild-type human aldolase B and the Val(337) variant enzyme were expres sed in Escherichia coli. The specific activities of the wild-type recombina nt enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosph ate (FBP) and fructose l-phosphate (F-I-P) as substrates with Michaelis con stants of 4 mu M and 2.4 mM respectively. The specific activities of purifi ed tetrameric Val(337) aldolase B, which affects an invariant residue in th e C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-I -P as substrates respectively; the corresponding Michaelis constants were 2 2 mu M and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val(337) variant enzymes respectively. The Val(337) mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val(337) protein had a distinct thermal denaturation profi le with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinit y and activity towards its specific F-1-P substrate and maintained apprecia ble activity towards FBP, In contrast, fluorescence studies of the mutant s howed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These fi ndings in a rare but widespread naturally occurring mutant implicate the C- terminus in the activity of human aldolase B towards its specific substrate s and demonstrate its role in maintaining the overall stability of the enzy me tetramer.