The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal.
To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia. grechkin@mail.knc.ru
Alexander N
AN
Grechkin
Mats
M
Hamberg
D000080:Acetals / Q000737:chemistry / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006207:Half-Life; D008042:Linolenic Acids / Q000737:chemistry / Q000378:metabolism; D008054:Lipid Peroxides / Q000737:chemistry / Q000378:metabolism; D031647:Psidium; D014297:Trimethylsilyl Compounds / Q000737:chemistry
C117640:12-(1',3'-hexadienyloxy)-9,11-dodecadienoic acid; D000080:Acetals; D005231:Fatty Acids, Unsaturated; D008042:Linolenic Acids; D008054:Lipid Peroxides; D014297:Trimethylsilyl Compounds; C057375:13-hydroperoxy-9,11,15-octadecatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase