Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase.
We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.
Govindasamy
Lakshmanan
L
Biochemistry and Molecular Biology, University of Florida and The McKnight Brain Institute, 1600 Archer Rd., Gainesville, FL 32610, USA.
Kukar
Thomas
T
Lian
Wei
W
Pedersen
Brenda
B
Gu
Yunrong
Y
Agbandje-McKenna
Mavis
M
Jin
Shouguang
S
McKenna
Robert
R
Wu
Donghai
D
D001665:Binding Sites; D002331:Carnitine / Q000737:chemistry* / Q000378:metabolism; D002332:Carnitine O-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D055672:Static Electricity
D002332:Carnitine O-Acetyltransferase; D002331:Carnitine