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.