Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation.
A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450(cin) (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450(cam) (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K(m) values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K(m) values reflect a decrease in the camphor binding affinity. Non-productive H(2)O(2) generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450(cam) and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H(2)O(2) and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.
Kim
Donghak
D
Department of Pharmaceutical Chemistry, University of California, San Francisco, Genentech Hall, N572D, 600 16th Street, San Francisco, CA 94158-2517, USA.
Heo
Yong-Seok
YS
Ortiz de Montellano
Paul R
PR
D000595:Amino Acid Sequence; D001483:Base Sequence; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D017931:DNA Primers; D008401:Gas Chromatography-Mass Spectrometry; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009243:NAD; D006861:Hydrogen Peroxide; D019475:Camphor 5-Monooxygenase