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  • Title: Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337-->Val) in fructose intolerance.
    Author: Rellos P, Ali M, Vidailhet M, Sygusch J, Cox TM.
    Journal: Biochem J; 1999 May 15; 340 ( Pt 1)(Pt 1):321-7. PubMed ID: 10229688.
    Abstract:
    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 Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer.
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