Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy.
Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid). P450SP(alpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450BS(beta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively. Using deuterium-substituted fatty acids ([2,2-d2]-myristic acid and d27-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated. In the P450SP(alpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on Vmax (DV = 4.1) when [2,2-d2]-myristic acid was used, suggesting that an isotopically sensitive step involving the alpha-hydrogen of the fatty acid is present in the catalytic cycle. On the other hand, D(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450. The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation. A similar kinetic isotope effect was observed [DV = 4.9, D(V/K) approximately 1] for the P450BS(beta) reaction, when d27-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases. The resonance Raman spectral data of P450BS(beta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed. On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450SP(alpha) and P450BS(beta).
Matsunaga
Isamu
I
Department of Virology and Department of Host Defense, Osaka City University, Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. matsunagai@med.osaka-cu.ac.jp
Yamada
Akari
A
Lee
Dong-Sun
DS
Obayashi
Eiji
E
Fujiwara
Nagatoshi
N
Kobayashi
Kazuo
K
Ogura
Hisashi
H
Shiro
Yoshitsugu
Y
D001412:Bacillus subtilis / Q000201:enzymology; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases; D008956:Models, Chemical; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D020578:Sphingomonas / Q000201:enzymology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C019081:fatty acid alpha-hydroxylase; D010544:Peroxidases; C437763:cytochrome P-450 CYP152A1 (Bacillus subtilis)