Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4.
Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E. coli fused to glutathione S-transferase. Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (delta2-27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (delta2-27); introduction of an alpha-helix-disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active-site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.
Schulze
J
J
Walther-Straub-Institut für Pharmakologie und Toxikologie der LMU, München, Germany.
Lehnerer
M
M
Lewis
D F
DF
Hlavica
P
P
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D008862:Microsomes, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D005982:Glutathione Transferase