HUMAN COPROPORPHYRINOGEN OXIDASE - BIOCHEMICAL-CHARACTERIZATION OF RECOMBINANT NORMAL AND R231W MUTATED ENZYMES EXPRESSED IN ESCHERICHIA-COLI AS SOLUBLE, CATALYTICALLY ACTIVE HOMODIMERS

To obtain recombinant human coproporphyrinogen oxidase (CPX), a cDNA f or the coding region of mature human CPX has been expressed in E. coli . CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage jus t preceding first amino acid of the CPX protein. The human CPX was fou nd to be in the soluble fraction. This previously unobtainable human h eme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration. Recombinant human CPX exhibit s homogeneous behavior during high performance liquid chromatography ( HPLC) and the N-terminal sequence, confirmed by protein sequencing, re vealed a single polypeptide chain. In its active form, human CPX is a homodimer. According to the hydrodynamic properties derived from analy tical ultracentrifugation, dimeric CPX has a nearly globular shape. Ad ditionally, naturally occurring Arg to Trp (R231W)-mutated CPX has bee n also expressed in E. coli and further characterized. The mutated enz yme has a K-m value of 0.55 mu M as compared to 0.30 mu M for the wild type. The catalytic efficiency (specificity constant, K-cat/K-m) of t he mutated CPX was four fold lower than wild-type enzyme. The activity measurement of the mutated:enzyme showed higher thermal sensitivity a s compared with wild type CPX. The measured pi for mutated CPX is 5.65 , compared to 6.40 for wild type. The pH optima for the mutated and wi ld-type protein are 6.6 and 6.8, respectively. The R231W mutation of C PX does not affect dimer formation and both normal and mutated CPX exh ibit identical sedimentation properties. The thermal denaturation of b oth wild type and mutant CPX was found to be irreversible. The mutated CPX contained a significant amount of tightly bound porphyrin copropo rphyrin. No metal association was found either in wild type or in muta ted CPX. The availability of the recombinant human CPX will aid in str uctural and mechanistic studies.