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

Department of Chemistry, Illinois State University, Normal, Illinois 61790-4160, USA

(RECEIVED October 26, 2006; FINAL REVISION November 27, 2006; ACCEPTED November 27, 2006)

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 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.

Keywords: coproporphyrinogen oxidase; enzyme kinetics; catalytic aspartate

Reprint requests to: Marjorie A. Jones, Department of Chemistry, Illinois State University, Normal, IL 61790-4160, USA; e-mail: majone3{at}ilstu.edu; fax: (309) 438-5538.

Abbreviations: CPO, coproporphyrinogen oxidase; copro'gen-III, coproporphyrinogen-III; proto'gen-IX, protoporphyrinogen-IX; hardero'gen, harderoporphyrinogen; meso'gen-VI, mesoporphyrinogen-VI.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062636907.

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