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.
Shoolingin-Jordan
P M
PM
Biochemistry and Molecular Biology, School of Biological Sciences, Southampton University, Biomedical Building, Bassett Crescent East, Southampton SO16 7PX, UK. pmsj@soton.ac.uk
Al-Dbass
A
A
McNeill
L A
LA
Sarwar
M
M
Butler
D
D
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011162:Porphobilinogen / Q000378:metabolism*
D003067:Coenzymes; C056776:dipyrromethane cofactor; D011162:Porphobilinogen; D011163:Hydroxymethylbilane Synthase