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Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation

Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation
Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation
Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.
dipyrromethane cofactor assembly, enzyme–intermediate complex, holo-deaminase formation, human porphobilinogen deaminase, tetrapyrolle formation
0300-5127
731-735
Shoolingin-Jordan, P.M.
ac0bf2cc-ee36-4b30-bcef-525cee2559f7
Al-Dbass, A.
e98af6ef-f26a-4dd6-b115-f4635b33298a
McNeill, L.A.
d4866b96-d99a-4fdf-af7e-71f383fee340
Sarwar, M.
5b6c7791-3f9b-43a2-a0a3-38e8289a8fde
Butler, D.
cc1f7fe2-ee46-45cf-bb99-edf377b1460e
Shoolingin-Jordan, P.M.
ac0bf2cc-ee36-4b30-bcef-525cee2559f7
Al-Dbass, A.
e98af6ef-f26a-4dd6-b115-f4635b33298a
McNeill, L.A.
d4866b96-d99a-4fdf-af7e-71f383fee340
Sarwar, M.
5b6c7791-3f9b-43a2-a0a3-38e8289a8fde
Butler, D.
cc1f7fe2-ee46-45cf-bb99-edf377b1460e

Shoolingin-Jordan, P.M., Al-Dbass, A., McNeill, L.A., Sarwar, M. and Butler, D. (2003) Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation. Biochemical Society Transactions, 31 (3), 731-735.

Record type: Article

Abstract

Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.

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More information

Published date: 1 June 2003
Keywords: dipyrromethane cofactor assembly, enzyme–intermediate complex, holo-deaminase formation, human porphobilinogen deaminase, tetrapyrolle formation

Identifiers

Local EPrints ID: 56753
URI: http://eprints.soton.ac.uk/id/eprint/56753
ISSN: 0300-5127
PURE UUID: 6435dbd7-4f22-475f-b835-ba0f34a62adf

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Date deposited: 06 Aug 2008
Last modified: 22 Jul 2022 21:07

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Contributors

Author: A. Al-Dbass
Author: L.A. McNeill
Author: M. Sarwar
Author: D. Butler

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