Symposium: Subcellular Compartmentation of Folate Metabolism Symposium on the Subcellular Compartmentation of Folate Metabolism1 CONRAD WAGNER Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 Department of Biochemistry, Vanderbilt University School of Medicine and Department of Veterans Affairs Medical Center, Nashville, TN 37232 This symposium was occasioned by an increased in It was therefore surprising to learn that purification terest in folate metabolism. This is a result of the re of the enzymes involved in one-carbon metabolism and cent discovery of the role of folate nutrition in the development of accurate methods to determine the size incidence of neural tube defects and the realization that of the one-carbon pools showed that the concentration folate deficiency is associated with hyperhomocysti- of the enzymes actually exceeds the concentration of nemia with the attendant increased risk of vascular the one-carbon folate pools. The folate pools consist of disease. When one undertakes to study the role of folate the polyglutamate derivatives of the folate coenzymes and one-carbon units in the cell, it soon becomes clear that have dissociation constants in the 100 nmol/L that metabolism is compartmentalized between the cy- range. These two facts suggest that in the cell most one-carbon derivatives of folate are present bound to tosol and the mitochondria. This is because almost all enzymes or other folate-binding proteins. The concen the folate in the cell is distributed almost equally be tween the two compartments. There are also mito- tration of the free pools of one-carbon folate coenzymes chondrial and cytosolic isoforms of the same enzymes are therefore in the low nmol/L range. This raises the question of whether the rate of one-carbon metabolism and the question arises as to the reasons for this redun is actually controlled by the rate of diffusion of sub dancy. See Figure 1. strates and products from these enzymes or whether The first speaker was Verne Schirch of the Depart there is some other explanation for the transfer of one- ment of Biochemistry of Virginia Commonwealth Uni versity. Schirch discussed the concept that compart- carbon units through a specific metabolic pathway. The concept of organized clusters of enzymes in mentation of folate metabolism may be present even metabolic pathways has been gaining acceptance (Srere within the cytosol itself. The role of the folate coen- 1987). In an enzyme cluster, the products of individual zymes is to carry one-carbon units and within the cyto enzyme catalyzed reactions are transferred directly to sol the folate coenzymes play a role in three major the next enzyme and act as substrates without diffusing biosynthetic pathways (Schirch and Strong 1989). into and out of the solvent. Such transfer of metabolites These are as follows: Î)the pathway of purine biosyn between enzymes is called channeling. Schirch has pu thesis in which folate provides a one-carbon unit at rified and studied the structural and kinetic properties two different steps, 2) the pathway of thymidylate bio of five folate-requiring enzymes that are involved in the synthesis in which a one-carbon unit is transferred to metabolism of 10-formyltetrahydrofoíate. This form of deoxyuridylic acid from 5,10-methylene tetrahydrofo- the coenzyme serves as the donor for carbons 2 and 8 late and simultaneously reduced to form the methyl in purine ring biosynthesis. The first of these enzymes group of thymidylic acid and 3) the formation of methi- is serine hydroxymethyltransferase, which uses the onine from homocysteine and 5-methyltetrahydrofo- third carbon of serine to generate 5,10-methylenetet- late in a vitamin B-12-dependent reaction. It has usu ally been assumed that the concentration of the re duced folates used as substrates for these reactions are 1A summary of manuscripts presented in the Symposium: "Sub- cellular Compartmentation of Folate Metabolism" given at the Ex greater than the concentration of the enzymes (which perimental Biology '95 meeting, Atlanta, GA, on April 10, 1995. This carry out a catalytic function) and that all enzymes symposium was sponsored by the American Institute of Nutrition. that use the same folate one-carbon derivative compete Guest editor for the symposium publication was Conrad Wagner, for a common pool. Vanderbilt University School of Medicine, Nashville, TN. 0022-3166/96 $3.00 ©1996 American Institute of Nutrition. J. Nutr. 126: 1228S-1234S, 1996. 1228S FOLATE METABOLISM 1229S ÇytoplMm Mitochondria l l » Folate -»•DHF-*•THF THF serine "rco.ine 14 14 12 glycine • glycine 6,10-methylene-THF 5,10-methylene-THF Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 a^—^- NADP* NADP* C02.NH3 i—^ NADPH NADPH 5,10-methenyl-THF 5,10-me theny 1-THF THF j 4 l' J-J •ormyl-THF-ÕÕNADP* punnes ^~ 10-fonnyl-THF lO-fonnyl ADP + Pi ADP + P 8 formate «*- •*•formate FIGURE 1 Compartmentation of folate one-carbon metabolism. The numbers refer to enzymatic reactions. They are as follows: 1, dihydrofolate reductase; 2, thymidylate synthase,- 3, 5,10-methylenetetrahydrofolate dehydrogenase,- 4, 5,10-methe- nyltetrahydrofoíate cyclohydrolase; 7,10-formyltetrahydrofoíate dehydrogenase; 8,10-formyltetrahydrofoíate synthase; 9, meth- ionyl-t-RNA formyltransferase; 12, sarcosine dehydrogenase; 13, glycine cleavage enzyme system; 14, serine hydroxymethyltran- sferase; 19, 5,10-methylenetetrahydrofolate reducíase.The activities carried out by reactions 3, 4, 7, 8 and 14 are found in both the cytosoplasm and the mitochondria. rahydrofolate (Schirch 1982). This folate is then con 10-formyltetrahydrof oíatesynthetase (L.Schirch, unpub verted to 10-formyltetrahydrof oíateby the methylene- lished results). In each of these reactions the channeling tetrahydrofolate dehydrogenase and methylenetetrahy- was only efficient for the polyglutamate forms of the drofolate cyclohydrolase activities of the trifunctional coenzyme. Although some evidence for physical associa enzyme Crtetrahydrofolate synthase (Schirch 1978). A tion between serine hydroxymethyltransferase and the third activity of this trifunctional enzyme is 10-formyl 10-formyltetrahydrof oíate dehydrogenase and synthe tetrahydrof oíatesynthetase, which forms 10-formyltet- tase enzymes has been obtained, no clear evidence of rahydrofolate directly from formate in an ATP-depen- how these enzymes might form a cluster has yet been dent reaction. The fifth enzyme purified and studied determined. One significant advantage of channeling of was 10-formyltetrahydrof oíatedehydrogenase (Schirch the reduced folate coenzymes in these reactions is the et al. 1994). This abundant liver enzyme was shown to known increased stability of the enzyme-bound coen bind tetrahydrofolate very tightly by Cook and Wagner zyme. (1982). Together, these five enzymes form two meta The second speaker was Donald W. Home of the bolic cycles that interconvert the third carbon of serine Department of Biochemistry at Vanderbilt University and formate. In rabbit liver the concentration of folate and the Veterans Administration Medical Center in binding sites on these five enzymes is ~30 fimol/L. Nashville. Home addressed the Compartmentation of These enzymes were purified and combined in vitro to folate-linked enzymes and coenzymes and also the is see if they form an enzyme cluster that channels the sue of how folate is transported into the mitochondria. reduced folate coenzymes. It is known that folate-linked enzymes are present Using a variety of kinetic methods evidence was pre in liver mainly in the cytosolic and mitochondrial frac sented that the tetrahydrofolate coenzymes are chan tions (Wagner 1982), and it has been shown that the neled between several of these enzymes. These include folate coenzymes are present also mainly in these two the following: methylenetetrahydrofolate dehydrogenase compartments (Cook and Blair 1979). Furthermore, in and methenyltetrahydrofoíate cyclohydrolase (Schirch vitamin B-12 deficiency induced either by dietary ma 1978); serine hydroxymethyltransferase and 10-formyl- nipulation or by nitrous oxide it is known that folate tetrahydrofolate synthetase (Strong and Schirch 1989); coenzymes accumulate as the 5-methyl derivative at serine hydroxymethyltransferase and 10-formyl tetrahy the expense of other reduced folates. This is called the drofolate dehydrogenase (L. Schirch, unpublished re methyl-trap hypothesis. It occurs because the B-12- sults); and methenyltetrahydrofolate cyclohydrolase and dependent enzyme, methionine synthase (which uses 1230S SUPPLEMENT 5-methyltetrahydrofolate to methylate homocysteine the tetrahydrofolate to generate 10-formyltetrahydrofo- and regenerate methionine and tetrahydrofolate), is in late, which may be oxidized via a mitochondrial ana hibited because of lack of vitamin B-12 or inactivation logue of 10-formyltetrahydrof oíatedehydrogenase. of the enzyme by nitrous oxide and because the en Because Home and his colleagues knew from their zyme, 5,10-methylenetetrahydrofolate reducíase(which previous studies that folylpolyglutamates could not synthesizes 5-methyltetrahydrofolate) is irreversible in readily cross the mitochondrial membrane, they asked vivo (Shane and Stokstad 1985). These facts led Home the question of how folates get into the mitochondria. to wonder whether cytosolic or mitochondrial folates An earlier report stated that only the oxidized folie