title: 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 Gly346-->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 Gly346Ser 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 kcat 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 degrees 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.
authors.affiliation: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, I-80131 Napoli, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Esposito
authors.forename: Luigi
authors.initials: L
authors.lastname: Vitagliano
authors.forename: Paola
authors.initials: P
authors.lastname: Costanzo
authors.forename: Loredana
authors.initials: L
authors.lastname: Borrelli
authors.forename: Rita
authors.initials: R
authors.lastname: Barone
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Pavone
authors.forename: Paola
authors.initials: P
authors.lastname: Izzo
authors.forename: Adriana
authors.initials: A
authors.lastname: Zagari
authors.forename: Francesco
authors.initials: F
authors.lastname: Salvatore
mesh_terms: D019943:Amino Acid Substitution; D000745:Anemia, Hemolytic, Congenital / Q000235:genetics*; D001176:Arthrogryposis / Q000235:genetics*; D002942:Circular Dichroism; D003062:Codon; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*; D005998:Glycine / Q000737:chemistry; D006579:Heterozygote; D006801:Humans; D007223:Infant; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D008970:Molecular Weight; D018908:Muscle Weakness / Q000151:congenital / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009243:NAD / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D009243:NAD; D005634:Fructose-Bisphosphate Aldolase; D005998:Glycine