The effects of various @entity20179 ( @entity20179 ) mutations responsible for @entity11160 ( @entity11160 ), the roles of the @entity369 -59 residue and the glycines in the conserved @entity1785 binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant @entity1 @entity20179 and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced @entity20179 activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind @entity1785 . The positive charge at @entity369 -59 appears to be directly involved in catalysis and not in @entity1785 -cofactor binding alone. The K(m)s for the @entity369 -59 mutants suggested a substrate-binding problem. T(1/2) indicated that @entity369 -59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that @entity369 -59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of @entity2228 in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for @entity70403 .

Kinetic and physical characterisation of recombinant wild-type and mutant XXXX protoporphyrinogen oxidases.

@entity1 :: ('9606', 'Species') :: ['human'] @entity70403 :: ('p|SUB|R|168|C', 'ProteinMutation') :: ['R168C'] @entity1785 :: ('MESH:C024132', 'Chemical') :: ['flavin'] @entity20179 :: ('5498', 'Gene') :: ['PPOX', 'protoporphyrinogen oxidase'] @entity2228 :: ('CHEBI:57305', 'Chemical') :: ['glycine'] @entity11160 :: ('MESH:D046350', 'Disease') :: ['VP', 'variegate porphyria'] @entity369 :: ('CHEBI:29016', 'Chemical') :: ['arginine']
@entity1 :: (9606,Species) :: ['human']
