The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies.
We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.
Hsu
L C
LC
Institute of Molecular Biology, Academia Sinica, Nankang and the Graduate Institute of the Cell and Molecular Biology, Taipei Medical College, Taipei, Taiwan, Republic of China.
Hsu
N C
NC
Guzova
J A
JA
Guzov
V M
VM
Chang
S F
SF
Chung
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013255:Steroid 21-Hydroxylase
D011994:Recombinant Proteins; D007532:Isoleucine; D003577:Cytochrome P-450 Enzyme System; D013255:Steroid 21-Hydroxylase