Study on Human 5-Aminolaevulinate Synthase and the Molecular basis for Pyridoxine Responsive X-Linked Sideroblastic Anaemia
Study on Human 5-Aminolaevulinate Synthase and the Molecular basis for Pyridoxine Responsive X-Linked Sideroblastic Anaemia
5-Aminolaevulinic acid is the first precursor of haem biosynthesis and is formed by the condensation between glycine and succinyl-CoA in a reaction catalysed by 5-aminolaevulinic acid synthase (ALAS). Pyridoxal 5'-phosphate (PLP) is required as a coenzyme. The enzyme is confined to animals, fungi and some bacteria.
The results reported in this thesis show that recombinant human erythrocyte 5-aminolaevulinic acid synthase (ALAS2), expressed as a His tagged protein in Escherichia coli, is active in vitro with a final specific activity of 18μmole/mg/hr. Steady state kinetics analysis of ALAS2 revealed a Km for glycine of 13.9 mM and a Km value for the second substrate, succinyl CoA, of 0.8μM. The reaction catalysed by ALAS2 was followed by UV/visible spectroscopy. The results showed that the enzyme binds to the coenzyme pyridoxal 5'-phosphate via an aldimine linkage. Two mutant forms of human ALAS2, Thr388Ser and Arg452His, both of which lead to pyridoxine responsive X-linked sideroblastic anaemias, were isolated and characterised by kinetic and spectroscopic techniques. Another mutant, Arg517Cys, that causes a refractory form of sideroblastic anaemia was also isolated and characterised by kinetic and spectroscopic analysis. Arginine 517 is implicated in binding the carboxyle group of the substrate glycine. In addition, the arginine 517 residue was mutated to leucine and lysine. Only the Arg517Lys mutant showed activity (5%), the other two mutants were being completely inactive.
The similarity in the main-chain folds between ALAS2 and 2-amino-3-oxononanoate synthase (AONS), a closely related PLP-dependent enzyme, permitted the constriction of a model structure for human ALAS2, using the sequence alignment of AONS as a template. The location of human mutations in the model structure of ALAS2 provides a rational explanation for many of the physiological effects of pyridoxine therapy.
University of Southampton
Aldaihan, Sooad K
0a06306f-a60f-4a66-aacc-f52e3ab35c19
2001
Aldaihan, Sooad K
0a06306f-a60f-4a66-aacc-f52e3ab35c19
Aldaihan, Sooad K
(2001)
Study on Human 5-Aminolaevulinate Synthase and the Molecular basis for Pyridoxine Responsive X-Linked Sideroblastic Anaemia.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
5-Aminolaevulinic acid is the first precursor of haem biosynthesis and is formed by the condensation between glycine and succinyl-CoA in a reaction catalysed by 5-aminolaevulinic acid synthase (ALAS). Pyridoxal 5'-phosphate (PLP) is required as a coenzyme. The enzyme is confined to animals, fungi and some bacteria.
The results reported in this thesis show that recombinant human erythrocyte 5-aminolaevulinic acid synthase (ALAS2), expressed as a His tagged protein in Escherichia coli, is active in vitro with a final specific activity of 18μmole/mg/hr. Steady state kinetics analysis of ALAS2 revealed a Km for glycine of 13.9 mM and a Km value for the second substrate, succinyl CoA, of 0.8μM. The reaction catalysed by ALAS2 was followed by UV/visible spectroscopy. The results showed that the enzyme binds to the coenzyme pyridoxal 5'-phosphate via an aldimine linkage. Two mutant forms of human ALAS2, Thr388Ser and Arg452His, both of which lead to pyridoxine responsive X-linked sideroblastic anaemias, were isolated and characterised by kinetic and spectroscopic techniques. Another mutant, Arg517Cys, that causes a refractory form of sideroblastic anaemia was also isolated and characterised by kinetic and spectroscopic analysis. Arginine 517 is implicated in binding the carboxyle group of the substrate glycine. In addition, the arginine 517 residue was mutated to leucine and lysine. Only the Arg517Lys mutant showed activity (5%), the other two mutants were being completely inactive.
The similarity in the main-chain folds between ALAS2 and 2-amino-3-oxononanoate synthase (AONS), a closely related PLP-dependent enzyme, permitted the constriction of a model structure for human ALAS2, using the sequence alignment of AONS as a template. The location of human mutations in the model structure of ALAS2 provides a rational explanation for many of the physiological effects of pyridoxine therapy.
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Published date: 2001
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Local EPrints ID: 464472
URI: http://eprints.soton.ac.uk/id/eprint/464472
PURE UUID: 4c96e826-3e9c-4061-b4cf-b56f7fa15134
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Date deposited: 04 Jul 2022 23:40
Last modified: 16 Mar 2024 19:32
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Sooad K Aldaihan
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