Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12202/2880
Title: STUDIES ON TWO ENZYMES IN THE CARNITINE BIOSYNTHETIC PATHWAY: 6-N-TRIMETHYL-L-LYSINE HYDROXYLASE AND SERINE TRANSHYDROXYMETHYLASE
Authors: STEIN, ROLAND WINTHROP
Keywords: Biology.
Issue Date: 1983
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 44-07, Section: B, page: 2050.
Abstract: L-Carnitine has an important function in facilitating the transport of long chain fatty acid acyl units across the inner mitochondrial membrane. Its pathway of biosynthesis in mammals from 6-N-trimethyl-L-lysine occurs via hydroxylation to yield 3-hydroxy-6-N-trimethyl-L-lysine, cleavage between carbons 2 and 3 to yield and 4-N-trimethylaminobutyraldehyde, oxidation of the latter to 4-N-trimethylaminobutyric acid, and finally hydroxylation at carbon 3 to yield L-carnitine. My studies delineated some of the features of 6-N-trimethyl-L-lysine hydroxylation in crude extracts of rat kidney homogenates, and compared the kinetic properties of this activity to homogenates from rat liver, skeletal muscle and heart tissues. In addition, the enzymes responsible for 3-hydroxyl-6-N-trimethyl-L-lysine cleavage in rat were purified, i.e. cytoplasmic and mitochondrial serine transhydroxymethylase (EC 2.1.2.1).;A tritium release assay for 6-N-trimethyl-L-lysine hydroxylase with chemically synthesized {lcub}3-('3)H{rcub} 6-N-trimethyl-D,L-lysine was developed. Utilizing this assay system, hydroxylation of 6-N-trimethyl-L-lysine to 3-hydroxy-6-N-trimethyl-L-lysine for several rat tissues has been examined and compared. The kidney enzyme was shown to require molecular oxygen and (alpha)-ketoglutarate as co-substrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K(,m) values were obtained for substrate, co-substrates, and co-factors. In view of similar kinetic parameters among the several hydroxylases examined, and the observation that the level of hydroxylase activity of skeletal muscle is comparable to that of heart, liver and kidney, skeletal muscle appears to be the most important tissue for hydroxylating 6-N-trimethyl-L-lysine for L-carnitine biosynthesis in the rat.;Specifically radiolabelled 3-hydroxy-6-N-trimethyl-D,L-lysine was chemically synthesized and used to assay the 3-hydroxy-6-N-trimethyl-L-lysine cleavage. 4-N-Trimethylaminobutyraldehyde, 4-N-trimethylaminobutyrate and L-carnitine were found during the metabolism of 3-hydroxy-6-N-trimethyl-L-lysine with liver extracts. This was the first demonstration of 4-N-trimethylaminobutyraldehyde accumulating during the biosynthesis of L-carnitine. Results of studies from the purification of 3-hydroxy-6-N-trimethyl-L-lysine aldolase in rat liver suggested that the activity is associated with both cytoplasmic and mitochondrial serine transhydroxymethylase. The activity of cytoplasmic serine transhydroxymethylase with 3-hydroxy-6-N-trimethyl-D,L-lysine, however, was 23 times greater than that of the mitochondrial enzyme. These results indicated that the cytoplasmic serine transhydroxymethylase functions in cleaving 3-hydroxy-6-N-trimethyl-L-lysine to 4-N-trimethylaminobutyraldehyde for L-carnitine biosynthesis in addition to its established role in one-carbon unit metabolism.
URI: https://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:8326719
https://hdl.handle.net/20.500.12202/2880
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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