title: Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase.
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.
authors.affiliation: Department of Chemistry, Illinois State University, Normal, Illinois 61790-4160, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Stephenson
authors.forename: Julie A
authors.initials: JA
authors.lastname: Stacey
authors.forename: Justin B
authors.initials: JB
authors.lastname: Morgenthaler
authors.forename: Jon A
authors.initials: JA
authors.lastname: Friesen
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Lash
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Jones
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry* / Q000235:genetics; D003305:Coproporphyrinogens / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008652:Mesoporphyrins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011165:Porphyrinogens / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D003305:Coproporphyrinogens; D008652:Mesoporphyrins; D011165:Porphyrinogens; D011994:Recombinant Proteins; D001224:Aspartic Acid; C018563:harderoporphyrinogen; D001120:Arginine; D003304:Coproporphyrinogen Oxidase; D000409:Alanine