Human aldolase A natural mutants: Relationship between flexibility of the C-terminal region and enzyme function

Abstract

We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly 346 → Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346 → Ser mutant, and the Glu206 → Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346 → Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206 → Lys mutant had a Km approx. 2-fold higher than that of both the Gly346 → Ser mutant and the wild-type enzyme, and a k cat value 40 % less than the wild-type. The Gly346 → Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6-7°C higher than that of the Glu206 → Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206 → Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site.