Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases
| Autor: | Anne V. Corrigall, Horst H. Klump, P.N. Meissner, Ralph E. Kirsch, Mbulelo H. Maneli, Lester M. Davids |
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| Rok vydání: | 2003 |
| Předmět: |
Oxidoreductases Acting on CH-CH Group Donors
Protein Denaturation Hot Temperature Variegate porphyria Mutant Mutation Missense Biophysics Flavin group Biochemistry Analytical Chemistry Mitochondrial Proteins Structure-Activity Relationship medicine Humans Protoporphyrinogen Oxidase Enzyme Inhibitors Binding site Molecular Biology Cofactor binding Flavoproteins biology Chemistry Wild type Active site medicine.disease Recombinant Proteins Kinetics Mutation Flavin-Adenine Dinucleotide biology.protein Protoporphyrinogen oxidase Oxidoreductases |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1650:10-21 |
| ISSN: | 1570-9639 |
| DOI: | 10.1016/s1570-9639(03)00186-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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