Assignment of endogenous substrates to enzymes by global metabolite profiling.
Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.
Saghatelian
Alan
A
The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Trauger
Sunia A
SA
Want
Elizabeth J
EJ
Hawkins
Edward G
EG
Siuzdak
Gary
G
Cravatt
Benjamin F
BF
D000581:Amidohydrolases / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D004983:Ethanolamines / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D051379:Mice; D018345:Mice, Knockout; D011237:Predictive Value of Tests; D013116:Spinal Cord / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity; D013654:Taurine / Q000378:metabolism; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D004983:Ethanolamines; D005227:Fatty Acids; D013654:Taurine; D000099252:Fatty Acid Amide Hydrolases; C022203:N-acylethanolamines