Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases.
The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-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 arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine 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 R168C.
Maneli
Mbulelo H
MH
Lennox Eales Porphyria Laboratories, MRC/UCT Liver Research Centre, Department of Medicine, University of Cape Town Medical School, K-floor, Old GSH Main Building, Observatory 7925, South Africa.
Corrigall
Anne V
AV
Klump
Horst H
HH
Davids
Lester M
LM
Kirsch
Ralph E
RE
Meissner
Peter N
PN
D004791:Enzyme Inhibitors / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D024101:Mitochondrial Proteins; D009154:Mutation; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011489:Protein Denaturation / Q000502:physiology; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D005420:Flavoproteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase