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

Gabriella Esposito, Luigi Vitagliano, Paola Costanzo, Loredana Borrelli, Rita Barone, Lorenzo Pavone, Paola Izzo, Adriana Zagari, Francesco Salvatore

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)51-56
Number of pages6
JournalBiochemical Journal
Volume380
Issue number1
DOIs
Publication statusPublished - May 15 2004

Fingerprint

Fructose-Bisphosphate Aldolase
Enzymes
Molecular graphics
Substitution reactions
Genes
Arthrogryposis
Mutation
Fructose
Hemolytic Anemia
Glycine
Serine
Freezing
Sequence Analysis
Melting point
Conformations
Catalytic Domain
Cats
Complementary DNA
Parturition
Rabbits

Keywords

  • Aldolase A
  • Aldolase A gene mutation
  • Aldolase A mutant expression
  • Fructose 1,6-bisphosphate
  • Molecular modelling

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human aldolase A natural mutants : Relationship between flexibility of the C-terminal region and enzyme function. / Esposito, Gabriella; Vitagliano, Luigi; Costanzo, Paola; Borrelli, Loredana; Barone, Rita; Pavone, Lorenzo; Izzo, Paola; Zagari, Adriana; Salvatore, Francesco.

In: Biochemical Journal, Vol. 380, No. 1, 15.05.2004, p. 51-56.

Research output: Contribution to journalArticle

Esposito, G, Vitagliano, L, Costanzo, P, Borrelli, L, Barone, R, Pavone, L, Izzo, P, Zagari, A & Salvatore, F 2004, 'Human aldolase A natural mutants: Relationship between flexibility of the C-terminal region and enzyme function', Biochemical Journal, vol. 380, no. 1, pp. 51-56. https://doi.org/10.1042/BJ20031941
Esposito, Gabriella ; Vitagliano, Luigi ; Costanzo, Paola ; Borrelli, Loredana ; Barone, Rita ; Pavone, Lorenzo ; Izzo, Paola ; Zagari, Adriana ; Salvatore, Francesco. / Human aldolase A natural mutants : Relationship between flexibility of the C-terminal region and enzyme function. In: Biochemical Journal. 2004 ; Vol. 380, No. 1. pp. 51-56.
@article{83c3eed08ec245dd91598b73d3bf2066,
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 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.",
keywords = "Aldolase A, Aldolase A gene mutation, Aldolase A mutant expression, Fructose 1,6-bisphosphate, Molecular modelling",
author = "Gabriella Esposito and Luigi Vitagliano and Paola Costanzo and Loredana Borrelli and Rita Barone and Lorenzo Pavone and Paola Izzo and Adriana Zagari and Francesco Salvatore",
year = "2004",
month = "5",
day = "15",
doi = "10.1042/BJ20031941",
language = "English",
volume = "380",
pages = "51--56",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

TY - JOUR

T1 - Human aldolase A natural mutants

T2 - Relationship between flexibility of the C-terminal region and enzyme function

AU - Esposito, Gabriella

AU - Vitagliano, Luigi

AU - Costanzo, Paola

AU - Borrelli, Loredana

AU - Barone, Rita

AU - Pavone, Lorenzo

AU - Izzo, Paola

AU - Zagari, Adriana

AU - Salvatore, Francesco

PY - 2004/5/15

Y1 - 2004/5/15

N2 - 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.

AB - 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.

KW - Aldolase A

KW - Aldolase A gene mutation

KW - Aldolase A mutant expression

KW - Fructose 1,6-bisphosphate

KW - Molecular modelling

UR - http://www.scopus.com/inward/record.url?scp=2642534395&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2642534395&partnerID=8YFLogxK

U2 - 10.1042/BJ20031941

DO - 10.1042/BJ20031941

M3 - Article

C2 - 14766013

AN - SCOPUS:2642534395

VL - 380

SP - 51

EP - 56

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -