Background: The catalytic contribution of four conserved histidines of human coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). Material/Methods: The wild-type and mutant enzyme forms were analyzed for their ability to utilize coproporphyrinogen-III, mesoporphvrinogen-VI, and harderoporphyrinogen as substrates. Results: Wild-type CPO had specific activities of 4.9 +/- 0.9 nmole product/min/mg for coproporphyrinogen-III, 1.7 +/- 0.7 nmole ptoduct/min/mg for mesoporphyrinogen-VI, and 5.1 +/- 1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent With all three substrates, but to varying degrees. The most affected Mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. Conclusions: Thus, His 158 of human CPO may have a role ill the active site, but none of the conserved histidine residues of human coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria.
Gitter, Shani J.; Cooper, Christopher L.; Frieson, Jon A.; Lash, Timothy D.; and Jones, Marjorie A., "Investigation of the catalytic and structural roles of conserved histidines of human coproporphyrinogen oxidase using site-directed mutagenesis" (2007). Faculty Publications – Chemistry. Paper 2.