Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase


Stephenson, JR; Stacey, JA; Morgenthaler, JB; Friesen, JA; Lash, TD; Jones, MA; (2007) Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase. Protein science, 16 (3). pp. 401-410. ISSN 0961-8368 DOI: https://doi.org/10.1110/ps.062636907

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Abstract

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 <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

Item Type: Article
Keywords: Alanine, Amino Acid Sequence, Amino Acid Substitution, Arginine, Aspartic Acid, Binding Sites, Catalysis, Coproporphyrinogen Oxidase, Coproporphyrinogens, Humans, Kinetics, Mesoporphyrins, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Porphyrinogens, Protein Binding, Recombinant Proteins, Sequence Homology, Amino Acid, Alanine, chemistry, Amino Acid Sequence, Amino Acid Substitution, Arginine, genetics, Aspartic Acid, genetics, Binding Sites, Catalysis, Coproporphyrinogen Oxidase, chemistry, genetics, Coproporphyrinogens, chemistry, Humans, Kinetics, Mesoporphyrins, chemistry, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Porphyrinogens, chemistry, Protein Binding, Recombinant Proteins, chemistry, genetics, Sequence Homology, Amino Acid
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
PubMed ID: 17242372
Web of Science ID: 244435900006
URI: http://researchonline.lshtm.ac.uk/id/eprint/8719

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