Carnitine biosynthesis via protein methylation

159 4 Tolbert, N. E. and Ryan, F. J. (1976) in COZ Meta(Burris. R. H. and bolism and Plant Productivity Black, CC., eds), pp. 141 -159, University Park Press, Baltimore, Md. 5 Shain, Y. and Gibbs, M. (1971) P/ant Physiol. 48, 325-330 6 Asami, S. and Akazawa, T. (1975) Planr Cell Physiol. 16,805-814 I Asami, S. and Akazawa, T. (1977) Biochemislry 16, in press 8 Kirk, M.R.and Heber, U.(1976)Planta 132,131141 9 Chollet, R. (1974) Arch. Biochem. Biophys. 163, 521-529 10 Lorimer, G. H., Andrews, T. J. and Tolbert, N. E. (1973) Biochemistry 12, 18-23 11 Bassham, J.A. and Kirk, M. (1973) PIanr Physiol. 52,407411 12 Eickenbusch, J. D., Scheibe, R. and Beck, E. (I 975) 2. Pflanzenphysiol. 75,375-380 13 Robinson, J.M. and Gibbs, M. (1974) Plant Physiol. 53,79&797 14 Andrews, T. J., Lorimer, G. H. and Tolbert, N. E. (1971) Biochemistry lo,47774782 15 Canvin, D.T., Lloyd, N.D.H., Fock, H. and Przybylla, K. (1976) in CO2 Metabolism and Plant Productivity (Burris, R. H. and Black, C. C., eds) pp. 161-176, University Park Press, Baltimore, Md. 16 Lorimer, G. H., Osmond, C. B. and Akazawa, T. (1976) Plant Physiol. 57 (suppl.), 6 17 Dimon, B. and Gerster, R. (1976) C.R. Acad. Sci. Ser. D 283,507-5 10 18 Halliwell, B. (1976) FEBS Lett. 64,266270 19 Bird, I.F., Cornelius, M. J., Keys, A. J. and Whittingham, C.P. (1972) Biochem. J. 128,191-192 20 Woo, K.C. and Osmond, C.B. (1976) Aust. J. Plant Physiol. 3,771-785 21 Grodzinski, B. and Butt, V. S. (1977) Planta 133, 261-266 22 Ellyard, P. W. and Gibbs, M. (1969) Plant Physiol. 44,1115-1121 23 Chollet, R. (1976) Plant Physiol. 57,237-240 24 Ehleringer, J. and BjGrkman, 0. (1977) Plant Physiol. 59,8&90 25 Nishimura, M., Graham, D. and Akazawa, T. (1975) Plant Physiol. 56,718-722 26 Bassham, J.A. and Kirk, M. (1962) Biochem. Biophys. Res. Commun. 9,376380 27 Badger, M.R. and Lorimer, G. H. (1976) Arch. Biochem. Biophys. 175,723%729 28 Chollet, R. and Anderson, L. L. (1976) Arch. Biothem. Biophys. 176,34+351 29 Brown, R. H. (1976) in CO1 Metabolism and Plant Productioity (Burris, R. H. and Black, C. C., eds), pp. 311-325, University Park Press, Baltimore, Md. 30 Keck, R. W. and Ogren, W. L. (1976) Plant Physiol. 58,552-555 31 Quebedeaux, B. and Chollet, R. (1977) PIant Physiol. 59,4244 32 Asami, S. and Akazawa, T. (1976) Planr Cell Physiol. 17, 1119-l 130

Carnitine biosynthesis via protein methylation Woon Ki Paik, Samuel Nochumson

and Sangduk

Kim

Carnitine, which is an acyl carrier within the mitochondrial membrane, is synthesized from .s-N-trimethyllysine in rat liver. Theformation of&-N-trimethyllysine resultsfrom enzymatic methylation ofprotein-lysine residues using S-adenosyl-L-methionine and subsequent degradation by hepatocyte lysosomes.

Protein methylation is one of several posttranslational modification reactions of polypeptides. Amino acid side chains such polypeptides. Amino acid side chains such as lysine, arginine, histidine and carboxyl groups are methylated with S-adenosyl-Lmethionine as the methyl donor by various methyltransferases specific to the amino acid residue involved [1,2]. Furthermore, the protein methylation reaction is ubiquitous in nature, as evidenced by the fact that various well-characterized and specialized proteins such as histone, myosin, actin, opsin, flagella protein, cytochrome c and ribosomal proteins of eukaryotic as well as prokaryotic organisms are found methylated in nature [2]. Unfortunately, however, a biochemical significance of this reaction has eluded us until now. In the following, we present the first example of an importance of the protein-lysine methylation reaction involving the biosynthesis of carnitine. Biosynthesis of carnitine It has been over seventy years since the fatty acyl group carrier molecule, carnitine, was first discovered in meat extract [3], yet its biosynthetic pathway has not yet been completely elucidated. The role of y-Ntrimethylaminobutyrate (y-butyrobetaine) as a precursor for carnitine in animals has long been established [4] along with methionine’s part as the source for methyl groups in this molecule, however only until a few years ago has the precursor of y-Ntrimethylaminobutyrate been identified. Thus, since &-N-trimethyllysine was first found in nature to be a constituent of protein [S], its close structural relationship to carnitine (Fig. 1) has led investigators to speculate correctly on its role as an intermediate in the carnitine biosynthetic pathway. Lysine was first demonstrated to be involved in carnitine biosynthesis by Horne, Tanphaichitr and Broquist, using a lysine auxotrophofNeurosporacrassagrownon a The authors are at the Fels Research Institute and Department of Biochemistry, Temple University School qf Medicine, Philadelphia, Pa. 19140, U.S.A.

carnitine free synthetic medium [S]. They showed that radioactivity derived from DL-[6-‘4C]lysine was incorporated into carnitine without dilution, however when either DL-[1-‘4C]lySine or DL-[2-t4C]lysine used, no radioactivity appeared in carnitine. The same results were found in an analogous experiment performed with rats fed a lysine-deficient diet, where radioactivity from DL-[6-‘4C]lysine was incorporated into carnitine but not from C-l or C-2 of lysine [7]. In addition, Cox and Hoppel [S] found that radioactive y-Ntrimethylaminobutyrate could be recovered from the urine of lysine-deficient rats injected with L-[U-14C]lysine, thus establishing the source of this already known precursor of carnitine. Havingestablishedthatlysinecan beconverted to carnitine, Broquist et al. demonstrated that a lysine auxotroph of Neurospora crassa was able to convert E-N-tri[methyl-‘4C]lysine to carnitine [9]. In rats kept on a lysine deficient diet, it was also found that injected .+N-tri[methyl-14C]lysine was metabolized to carnitine and that labeled y-N-trimethylaminobutyrate could be identified, suggesting a precursorproduct relationship [lo]. The transformation of &-N-trimethyllysine to carnitine seems to require at least four reactions based on the following evidence (outlined in Fig. 2). First, &-N-trimethyllysine is probably hydroxylated at carbon number 3 since Hoppel et al. [ 1l] have recently identified P-hydroxy+N-trimethyllysine in rat kidney and liver, and have shown that this compound can be further metabolized to carnitine. These data support a pathway postulated by Hochalter and Henderson who showed that rats administered both &-N-trimethyl-[l-‘4C]lysine and sodium benzoate contained radioactive hippuric acidin theirurine [ 121. The authors suggest that an aldolase reaction cleaving /?-hydroxy-s-N-trimethyllysine to yield glycine and the corresponding aldehyde is followed by oxidation of the aldehyde to yield ;q-Nmethylaminobutyrate. The hydroxylation of y-N-trimethylaminobutyrate to form carnitine has already been established [4]. Another possible route of carnitine bio-

TIBS - Ju!v 1977

160 Structural carnitine

similarity

of E-N-trimethyl-L-lysine

Relationship between protein methylation and carnitine

and

+

F: ~WM,

N .(CH&

CHz

CHz

I CHz I .

CHOH

CHz

CHz

CHz

COOH

An important link between protein methylation and carnitine biosynthesis stems from the work of LaBadie, Dunn and Aronson [14] who used chemically [methyl-‘4C]labeled asialo-fetuin as a source of c-N-trimethyllysine. When this modified protein was administered to rats, it quickly found its way to the hepatocyte lysosomes where active proteolytic enzymes rapidly catalyzed the production of free &-N-trimethyllysine. Carnitine was the major radioactive product detected in the extracts of rats carcass and liver within 3 hours after administering the methyl labeled fetuin. Approximately 35% of the E-Ntrimethyllysine found in the administered protein was converted to carnitine. Interestingly, the authors failed to detect carnitine formation from methyl labeled fetuin which contained only &-N-mono and E-Ndimethyllysine residues, even at a point 22 hours after its administration. This finding indicates that only &-N-trimethyllysine

I I

CH.NH2 COOH E-N-Trimethyllysine Figure

Carnitine

1

synthesis may involve the intermediate a-keto-e-N-trimethylhexanoic acid, which has &en identified as a metabolite of E-Ntrimethyllysine in Neurospora cruxsu by Villanueva and Lederer L-131.a-Keto-sN-trimethylhexanoic acid could be formed by the action of L-amino acid oxidase on E-N-trimethyllysine. Proposed

pathway

ofcarnitine

biosynthesis

residues in protein are of value for carnitine biosynthesis, whereas &-N-mono and &-N-dimethyllysine residues once freed by proteolysis do not appear to be further methylated to the trimethyl level and cannot be utilized for carnitine synthesis. The fate of the released &-N-mono and &-N-dimethyl-L-lysine may be salvaged as free lysine by the enzyme &-alkyllysinase [E-Alkyl-L-1ysine:OxygenOxidoreductase; EC 1.5.3.41 [IS], which oxidizes the methyl groups of these amino acids but not E-Ntrimethyl-L-lysine [ 163, to formaldehyde and return this essential amino acid to its unmethylatedform. This finding appears to be contrary to the case found for Neurospora crassa in which free lysine undergoesastepwisemethylationtothe trimethyl derivative [17], however it is also known that this organism contains e-N-trimethyllysine at the protein level [lS]. Thus, in mammalian species, protein methylation probably accounts for the major portion of &-N-trimethyllysine necessary for carnitine synthesis. The enzyme responsible for methylating

in mammals

&-M, I ‘=I I

CHz

CHz

CHz

CH2 I CHz I CHz

. ..CO-NH-C-CO-NH... H

. ..CO-NH-C-CO-NH... H

Protein

s-N-Trimethyllysine protein

C& Protein

methylase

III

.

CHz S-adenosyk-methionine

Proteolysis

CHz ,

I CHz CHz CH-NH2 COOH

lysine residue

in Free E-N-trimethyllysine

\ &CH3)3

&H3)3

CH*

CHz I C& l

CHOH

+

Hydroxylation

--

+ N-W93

?

?

L-Amino acid oxidase or transaminase (in N. crawa)

hH3)3

I ‘=z

I CHz

CH2

CH2

Aldolase

CH2

CH2

COOH

COOH

CHz I

CHz I

y-N-Trimethylamino butyric acid

CHOH

CHz

CH-NH~

c=o

COOH

COOH

Carnitine

CH2-NH2 COOH Glycine

Figure 2

B-Hydroxy-E-Ntrimethyllysine

a-Keto-s-N-trimethylaminohexanoic acid

161

TIBS - JU[V I977 protein-lysine has been designated protein methylase III [S-adenosylmethionine : protein-lysine methyltransferase; EC 2.1.1.431. This enzyme is found widely distributed throughout the various rat tissues [19]. Althoughmethylation ofthe lysine residues following synthesis of the protein probably serves a particular protein in achieving its functional properties, a secondary feature of this reaction is the production of E-Ntrimethyllysine, an amino acid which in mammals apparently cannot be synthesized in the free state. Since protein synthesis requires a large energy expenditure, we are not suggesting that some proteins are synthesized only for making s-N-trimethyllysine, but rather when considering protein catabolism, the cell can conveniently utilize the free carnitine precursor. Thus, this is a first example of a definite biological function for the protein methylation reaction. Acknowledgements This work was supported by research grants AM 09602 from the National Institute ofArthritis, Metabolism and Digestive Diseases, CA 10439 and CA 12226 from the National Cancer Institute, and GM 20594 from National Institute of General Medical Sciences. References 1 Paik, W. K. and Kim, S. (1971) Science 174,114 2 Paik, W. K. and Kim, S. (1975) in Adoance in Enzymology (Meister, A., ed.) John Wiley & Sons, New York, pp. 42-227 3 Gulewitsch, V.S. and Krimberg, R. (1905) Z. Physiol. Chem. 45,326 4 Lindstedt, G. and Lindstedt, L. (1961) Bioc~hrm. Biophys. Res. Commun. 6,3 19 5 Hempel, V.K., Lange, H.W. and Birkofer, L. (1968) Z. Naturforsch. 1, 37 6 Horne, D.W., Tanphaichitr, V. and Broquist, H.P. (1971) J. Biol. Chem. 246,4373 7 Tanphaichitr, V., Horne, D.W. and Broquist, H. P. (I 97 I ) J. Biol. Chem. 246, 6364 8 Cox, R.A. and Hoppel, CL. (1973) Biochem. .I. 136, 1075 9 Horne, D. W. and Broquist, H. P. (1973) .I. Biot. Chem. 248,217O 10 Cox, R.A. and Hoppel, C. L. (1973) Biochem. J. 136,1083 11 Hoppel, C. L., Novak, R. and Cox, R. A. (1976) Fed. Proc. 35,1478, Abt. 623 L. M. (1976) Bio12 Hochalter, J. B. and Henderson, them. Biophys. Res. Commun. 70,364 13 Villanueva, V.R. and Lederer, E. (1975) FEBS Lett. 52. 308 14 LaBadie, J.H., Dunn, W.A. and Aronson, N.N. (1976) Biochem. J. 160,85 15 Kim. S., Benoiton, L. and Paik. W.K. (1964) 1. Biol. Chem. 239,379O 16 Paik, W.K. and Kim, S., unpublished data. 17 Rebouche, C. J. and Broquist, H. P. (1976) J. Bacterial. 126, 1207 18 DeLange, R.J., Glazer, A.N. and Smith, E.L. (1969) J. Biol. Chem. 244,1385 19 Paik, W. K. and Kim, S. (1970) J. Biol. Gem. 245, 6010

Mechanisms of folate cofactors Stephen

J. Benkovic

and Charles

M. Tatum

Jr.*

Stereospecific isotopic substitution of hydrogen at the bridging carbon in 5,10-methylene tetrahydrofolate promises fiuther insight into stereochemical events at the active site qf several foiate requiring enzymes. Derivatives of tetrahydrofolate participate as cofactors in a variety of biologically important enzyme-catalyzed transformations including the biosynthesis of purines and pyrimidines, the metabolism of amino acids and the biosynthesis of methyl groups. A common feature of these reactions is the transfer of a one-carbon unit although at differing levels of oxidation. In this review we will consider only those reactions which utilize 5, lo-methylenetetrahydrofolate (CHz-Hd-folate) in which the one-carbon unit is effectively at the oxidation level of formaldehyde. Our intent is to examine the mechanisms of two of these transformations, the serineglycine interconversion and the biosynthesis of thymidylate, and particularly those aspects revealed through experiments designed to monitor the stereochemical events attendant with the reactions. Cofactor The structure of CHz-Hd-folate, 1, illustrates the basic pteroylglutamate structural unit common to the folate cofactors with the transferable one-carbon methylene unit bridging the 5-and lo-nitrogens. Poly-y-glutamyl conjugates also occur and are biologically active. There are two asymmetric centers in the parent molecule, one at C-6 in the tetrahydropyrazine ring and the second at the x-carbon of the gluta-

mate residue. The enzyme catalyzed reactions under discussion are stereospecilic for the (+),L-diastereomer in which the The authors are at the Department of Chemistry.The Pennsylvania State University, University Park, Pennsylvania 16802, U.S.A.

glutamate is in the L-configuration while the absolute configuration at C-6 is unknown [1,2]. The bridging methylene unit is a prochiral center, so that substitution of either hydrogen with a heavier isotope, for example, deuterium or tritium, converts this carbon to a third asymmetric center permitting one to follow the steric course of its transfer. Although the confactor is depicted in one of several possible conformations, all share the common feature that one face of the five-membered imidazolidine ring, the side bearing HA, is sterically more accessible than the other. For this discussion, let us assume that the absolute configuration of the molecule at C-6 is as drawn, since lacking this information we can discuss only relative stereochemical events. Serine-glycine interconversion A partial listing of the reactions catalyzed by the enzyme, serine hydroxymethylase from rabbit liver [3,4], indludes: L-serine + H4-folatee glycine + CHz-H4-folate r-methylserine

(1)

+ H4-folateti D-alanine

L-threonineeglycine

+ CH2-H4-folate

+ acetaldehyde

(2) (3)

The enzyme requires pyridoxal phosphate (PyP) [3], and considerable spectrophotometric evidence has been amassed implicating the formation of the respective amino acid-PyP imines as required steps in the above reactions. The PyP-glycine anion species is a presumed intermediate in the stereospecific replacement of -CHlOH and -CHOHCHj by -H at the pro-2S-position of glycine [5,6]. A comparison of I’,,,,, values indicates that serine reacts ten times more rapidly than either threonine or x-methylserine [7]. The reversible interconversion of serine and glycine has been demonstrated to occur in the absence of Hd-folate; but the rate of serine degradation and synthesis is “Present address: Department of Chemistry, Middlebury College, Middlebury, Vermont 05753, U.S.A.