Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase
| Autor: | Justin B. Morgenthaler, Julie A. Stacey, Marjorie A. Jones, Jon A. Friesen, Timothy D. Lash, Jason R. Stephenson |
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| Rok vydání: | 2007 |
| Předmět: |
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Molecular Arginine Stereochemistry Molecular Sequence Data Biochemistry Catalysis Article Coproporphyrinogen Oxidase Humans Amino Acid Sequence Enzyme kinetics Molecular Biology Oxidative decarboxylation Alanine chemistry.chemical_classification Aspartic Acid Binding Sites Sequence Homology Amino Acid biology Coproporphyrinogens Active site Substrate (chemistry) Porphyrinogens Recombinant Proteins Amino acid Kinetics Amino Acid Substitution Mesoporphyrins chemistry Mutagenesis Site-Directed biology.protein Protein Binding |
| Zdroj: | Protein Science. 16:401-410 |
| ISSN: | 1469-896X 0961-8368 |
| DOI: | 10.1110/ps.062636907 |
| Popis: | Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency |
| Databáze: | OpenAIRE |
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