Binding of 7-methoxy-4-(aminomethyl)-coumarin to wild-type and W128F mutant cytochrome P450 2D6 studied by time-resolved fluorescence spectroscopy.
Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128-->Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC.
Stortelder
Aike
A
Laser Centre VU, Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Keizers
Peter H J
PH
Oostenbrink
Chris
C
De Graaf
Chris
C
De Kruijf
Petra
P
Vermeulen
Nico P E
NP
Gooijer
Cees
C
Commandeur
Jan N M
JN
Van der Zwan
Gert
G
D019943:Amino Acid Substitution / Q000235:genetics*; D016880:Anisotropy; D003198:Computer Simulation; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
C120605:7-methoxy-4-(aminomethyl)coumarin; D003374:Coumarins; D019389:Cytochrome P-450 CYP2D6