Investigation of the catalytic and structural roles of conserved histidines of human coproporphyrinogen oxidase using site-directed mutagenesis.
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).


METHODS
The wild-type and mutant enzyme forms were analyzed for their ability to utilize coproporphyrinogen-III, mesoporphyrinogen-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 product/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, His158 of human CPO may have a role in 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
SJ
Department of Chemistry, Illinois State University, Normal, IL, USA.
Cooper
Christopher L
CL
Friesen
Jon A
JA
Jones
Marjorie A
MA
D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003305:Coproporphyrinogens / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D003305:Coproporphyrinogens; C471213:His-His-His-His-His-His; D050505:Mutant Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C028026:coproporphyrinogen III; D006639:Histidine; D003304:Coproporphyrinogen Oxidase