Topological role of cytochrome P450 2D6 active site residues.
Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.
van Waterschoot
Robert A B
RA
LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Keizers
Peter H J
PH
de Graaf
Chris
C
Vermeulen
Nico P E
NP
Tschirret-Guth
Richard A
RA
D019943:Amino Acid Substitution; D003198:Computer Simulation; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D019389:Cytochrome P-450 CYP2D6