Z

NAD 'Branched-ctiam CoA-SH a-keto acid dehydrogenase {TPP lipoamtde)

S-fi-isobutyrate

S-CoA

/CH\ HjC CH:i lsobutyryl*CaA

11-Methyl acyl-CoA dehydrogenase r FAD

FADH, cooh

3- h ydroxy i sobuty rate

CoA-SH H

-Hydroxy is obutyryl-CoA hydrolase

S-CoA I

Enoyi-CoA hydratase

S-CoA I

Melhylacrylylyl-CoA

3-Hydroxy-isobutyrate dehydrogenase

COOH

CH 1

Methylmalonate semialdehyde

NAD NADH CoA-SH CO,

Methylmalonic semialdehyde H3C dehydrogenase c* — -

Propionyl CoA

COOH C 0

H2 S-CoA

Succinyl CoA

Figure B.Ml Mcrabolisrn of L-vllrnf

Subsequent reaction are catalyzed bv 3-hydroXybutyryl-CoA dehydratase (crotonasc. EC4.2.1.55), 3-hydroxyisobutyryl-CoA hydrolase (EC3.1.2.4), 3-hydroxybutyrate dehydrogenase (EC1.1.1.1.31), and methylmalonic semialdehyde dehydrogenase (ECI.2.1.27). The product of the last reaction is propionyl-CoA. Conversion of propionyl-CoA in three additional steps to tiie Krebs cycle intermediate succinyl-CoA requires biotin and cobalamin.

While there is little transamination of Val in the liver due to the low activity (compared to muscle) of branched-chain amino acid aminotransferase (EC2.6.1.42). the potentially toxic product methacrylyl-coenzvme A is rapidly metabolized in liver due to the relatively high aeti\ ity of oxoglutarate dehydrogenase I funiguchi et al., 19%).

The concentration of the Val transamination product, alpha-kctoisovaleric acid, does not increase after a meal. Its concentration in muscle is only twice that of its hydrox-ylaled metabolite, aIpha-hydroxy isovaleric acid (H offer et at., 1993). This pattern differs from those resulting with the other two branched-chain amino acids: the concentrations of their transamination products increase after a meal and are hundredfold higher than the respective hydroxylated metabolites.

Storage

Most Val is bound in proteins, much of that in muscle which contains around 6(1 nig g protein. A 70 kg healthy adult might thus have reserves of about 360 g mobilizablc Val.

Excretion

Recovery of Val from glomerular filtrate in the proximal renal tubule closely resembles absorption from the small intestinal lumen. The sodium-driven transporter lis (Ax issar et al.. 2001) carries the bulk into the cell, with some help from the hetero-exchangcr 13AT1 b" (Chairoungdua et at., 1999), Another hetcroexchanger. LAT2 (SI.I 7AX) linked to the membrane glycoprotein 4F2lSLC3A2), completes the process by transporting Val across the basolater.il membrane in exchange for other neutral amino acids. Di- and tripeptides are taken up \ ia PepTl (SLCI5AI) and PepT2 (SLCI5A2), and cleaved in the tubular cell. Since most Val is reabsorbed, healthy people lose very little with urine. Losses with feces are also negligible as long as gastrointestinal function remains normal.

Most nitrogen from metabolized Val is excreted eventually into urine as urea. Regulation

Branched-chain alpha-keto acid dehydrogenase (EC'2.6.1.42) is the key regulatory enzyme for Val metabolism, which helps preserve this essential amino acid for protein synthesis during starvation. The enzyme is inactivated by a specific, calmodul independent kinase ([3-metbyl-2-oxobutanoate dehydrogenase (lipoamide)] kinase; EC2.7.1.U5) and activated by a specific phosphatase ([3-methyl-2-oxobutanOate dehydrogenase (lipoamide)]-phosphatasc; EC3.1.3.52). Branched-chain alpha-keto acid dehydrogenase is also inhibited by the products of the reaction it catalyzes, iso-valeryl-CoA. isobutyrvl-t'oA. and 2-methylbutyryl-CoA.

An intake imbalance with inadequacy of'one of the branched-chain amino acids (Val, leucine, or isoleucine) causes activation of orbiiofrontal cortex, frontal gyrus and thalamus and leads to the development of conditioned taste aversion (Gietzen and Magrum, 2001). Expression of several genes in response to inadequacy changes, but the mechanisms responsible for learning to balance branched-chain amino acid intake remain elusive.

Function

Energy fuel: Eventually, most Val is broken down providing about 5,3 kcal'g. A large percentage of ingested Val is oxidized soon after absorption. The main site of Val breakdown is skeletal muscle. Liver, intestines and other organs are responsible for much smaller percentages of Val utilization as an energy fuel.

Protein synthesis; Virtually ait proteins contain Val as pan of their specific sequence. Valine-tRNA ligase (EC6.1.1.9) is responsible for loading Val onto its specific tRNA. A lack ofVai. as of at) essential amino acids, slows protein synthesis. Like the other branched-chain amino acids. Val has anabolic effects on skeletal muscle (Anthony etal., 2001 >. The effect of L-lcucine is better understood, however.

Neurotransmitter metabolism: It has been suggested that Val and the other bran eh ed-chain amino acids promote nitrogen transfer between astrocytes and neurons in the brain. This BC'AA shuttle might be critical for the synthesis of the neurotransmitter L-glutamate (Hutson et al, 2(H) I).

References

Andresen BS, Christensen E. Corydon TJ. Bross P. Pilgaard B. Wanders RJ, Ruiter .IP, Simon sen H, Winter V. Knudsen I, Schroeder LP. Gregerscn N. Skovby F. Isolated 2-methyIbutyrylglycinuria caused by short branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and ev idence for distinct acyl-CoA dehydrogenases in isoleucinc and valine metabolism. Am J Hum Genet 20(H);67:1095 -103 Anthony JC, Anthony fG. Kimball SR, Jefferson LS Signaling pathways involved in translational control of protein synthesis in skeletal muscle by leucine. J \iilr 2001:131:856S-860S

Avissar NE. Ryan CK, Ganapathy V Sax HC. Na -dependent neutral amino acid trans-porler ATBo is a rabbit epithelial cell brush border protein. Am J Physiol Cell Physiol 2001 ;281 :C963-7(

Cctin 1. Amino acid inierconversions in the fctal-placental unit: the animal model and human studies in vivo. PecI Res 2001;49:148-53 Chairoungdua A, Segawa II, Kim JY. Miyamoto K. 11 ay a II, Fukui Y, Mizoguchi K, Ito H, Takcda E, Endou H. Kanai Y Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein,,/ Biol Client 1999:274:28845 8 Dancis J. I lutzler J, Tada K, Wada Y. Morikawa T. Arakawa T. I lypervalinemia: a defect in valine transamination. Pediatries 1967;39:813-17 Gietzen DW, Mag rum LJ. Molecular mechanisms in the brain involved in the anorexia of branched-chain amino acid deficiency. J Nutr 2001:851S 855S Hoffer LJ, FaveroffA. Robitaille L, Mamer OA. Reimer ML. Alpha-ketoand alpha-hydroxy branched-chain acid interrelationships in normal humans. J Nutr 1993:123:1513 21 Hutson SM. Lieth L. LaNoue KE. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J Nutr 2001; 131:846S-850S Jansson I". Amino acid transporters in the human placenta. Pediatr Res 2001:49:141 7 Matthews DL. Marano MA. Campbell KG. Splanchnic bed utilization of leucine and phenylalanine in humans. Un J Physiol I993;264:E109- 18

Roe CR. Cederbaum SP. Roe DS, Mardach R. Galmdo A. Sweet man I .. Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized defect in human valine metabolism. Mot Genet Met ah 1998;65:264-71 laniguchi K. Nonamt T. Nakao A. Harada A. Kurokawa T, Sugiyatna S. Fujilsuka N, Shimomura Y. Hutson SM. Harris RA. Takagi H. The valine catabolic pathway in human liver: effect of cirrhosis on enzyme activities. Hepatol 1996:24:1 395-S Telford EA. Moynihan l.M. Markham AE Lench NJ. Isolation and characterization of a cPNA encoding the precursor for a novel member of the acyl-CoA dehydrogenase gene family. Biochim BiophvsActa 1999; 1446:371 -6 Verrcy K Jack I)L. Paulsen IT. Saier Mil jr. Pfeilfer R. New glycoprotein-associated ammo acid transporters ./ Membrane Biol 1999;! 72:181 92 Young VR. Borgonh.i S. Nitrogen and amino acid requirements: the Massachusetts Institute ofTechnology amino acid requirement pattern../ Nutr2000; 130:1841S-49S

Isoleucine

The hydrophobic neutral amino acid L-isoleucine (2-amino-3-methy I valeric acid COOH

alpha-amino-bcta-methylvaleric acid 2-amino-3-methylpentanoic acid one-letter —¿H

code I; molecular weight 131 ) contains 10.7% nitrogen, '

LrH-i 1

Abbreviations / \

HjC CH^

BCAA branched-chain amino acids

CoA coenzyme A CH3

ETF electron transfer flavoprotein

FAD flavin adenine ¿¡nucleotide Figur« 8.61

lie L-isoleucine L-tsoicucme

LAT1 L-type amino acid transporter 1 (SLC7A5)

LAT2 L-type amino acid transporter 2 (SLC7A8)

RDA recommended dietary allowance

TPP thiamin pyrophosphate

Nutritional summary

Function: The essential amino acid L-isoleucine (tie) is needed for the synthesis of proteins. It is also an important energy fuel, especially in skeletal muscle: its breakdown requires thiamin, riboflavin, niacin, vitamin B6. vitamin BI2, pantothenate, biotin, lipoate. ubiquinone, magnesium, and iron.

Food sources: Dietary proteins from different sources all contain lie. Protein from dairy has a slightly higher content (6% of total protein) than protein from meats, or soy (around 5% of total protein), or grains (3-4"«). Dietary supplements containing crystalline lie, often in combination w ith other amino acids, are commercially available. Requirements: Adults are thought to require at least 23 mg/kg per day (Young and Botrgonha, 2000).

Deficiency: Prolonged lack of Me as of all essential amino acids or a lack of protein causes grow th failure, loss of muscle mass and organ damage. Excessive intake. Very high intake of protein and mixed amino acids (more than three times the RDA or 2.4 g kg) is thought to increase the risk of renal glomerular sclerosis and accelerate osteoporosis.

Dietary sources

Alt food proteins contain He. though the relative content varies slightly between different types of foods. Relatively rich sources are proteins from milk (60 mgg). egg (55 mgg). and soy (52 mg g). Intermediate contents are found in meats, such as chicken (49mgg). pork (47mg g), beef (45mg'g). and fish (46mgg). Lower contents are typical for grains such as corn (36mg g), wheat (39mgg). and slightly more in rice (43mg/g). Overall, the amount of protein consumed has greater impact on He intake than the source of the protein.

Digestion and absorption

Proteins are hydrolyzed by various enzymes; most of them arc derived from the stomach wall and pancreas.

The hydrogen ion peptide cotransporters 1 (PepTl. SLt'l 5A1) and toa lesser extent, 2 (PepT2. SLC15A2) mediate uptake of di- and tripeptides. including those containing Leu. Free f eu can cross the small intestinal bmsh border membrane via the sodium-driven transponer B® (identical with system B NÜB. Avissar et al.. 20011, sodium-dependent system L. and a specific sodium, leucine CO transporter, which is only partially characterized, as yet.

I ike many other neutral amino acids, lie can be exchanged (brother neutral amino acids across the brush border membrane in either direction b\ the rBAT (SLC3AI )-anchored amino acid transporter BATI/b° (SLC7A9. Chaíroungdua et al.. 1999),

The 4F2 (SLC3A2) glycoprotein-anchored transporter LAT2 (SLC7A8) can exchange lie across the basolateral membrane in both directions in exchange for other neutral amino acids.

Transport and cellular uptake

Blood circulation: lie is a ubiquitous component of proteins in blood and can be taken up by cells via mechanisms specific to the respective proteins. Free lie (plasma concentrations are around 65 (tmol I) enters cells mainly via system L, including the specifically identified heteroexchanger LATI (SLC7A5). The branched-chain amino acid transaminase (FC'2.6.1.42). which is located al the inner mitochondrial membrane, may function as an importer of branched-chain amino acids.

Materno-fetal transfer: He uptake across the microvillous membrane of the syncy-tiotrophoblast is mediated by LATI (Ritchie and Taylor, 2001). transport across the basal membrane proceeds via 1.AT2. both in exchange for other neutral amino acids

membrane membrane epithelium

Ftgur* £.62 Intestinal absorption of L-isoteucing membrane membrane epithelium

Ftgur* £.62 Intestinal absorption of L-isoteucing

(Jansson. 2001 }. The driving force for LATI/LAT2-mediated transport is the concentration gradient of small neutral amino acids (glycine, L-alanine. L-cysteine) established bv the sodium-dependent transport systems A and ASC.

Blood brain barrier: System I mediates lie transport aeross both sides of the neu-roendothelial cell layer. The molecular identity and location of the responsible trans-porter!s) remains unclear.

Metabolism

He breakdown to propionyl-CoA and aceiyt-CoA proceeds in six steps which are dependent on adequate supplies of riboflavin, pyridoxine, niacin, and pantothenate. Complete oxidation of propionyt-CoA and acctyl-CoA then also requires thiamin, cobalamin, biotin. and ubiquinone.

The pyridoxal 5'-phosphate (PLP)-dependent branchcd-chain amino acid aminotransferase 2 (EC2.it. 1.42) starts lie metabolism by moving its amino group to alpha-ketoglutarate. Another minor PLP-depcndent cn/ymc, vaIine-3-methyl-2-oxova I erate aminotransferase (EC2A1,32), can transfer the amino group to alpha- ketovalerate, thus reconstituting L-valinc (Dancisf/ci/,, I%7), The reverse reaction. which would reconstitute lie, might actually be more important

Branchcd-chain alpha-keto acid dehydrogenase (EC1.2.4.4) in the mitochondrial matrix acts on all three BCAAs. This large enzyme complex consists of multiple copies of three distinct subunits. The stibunit PI catalyzes the decarboxylation reaction with reduced coenzyme A as a cosubstrate. El itself is a heterodimerofan alpha chain with thiamin pyrophosphate (TPP) as a prosthetic group, and a beta chain. Subunit E2 anchors the lipoic aeid residue, which serves as an acceptor ("or the decarboxylated substrate, transfers it to acetyl-CoA. and reduces lipoamide to dihydroIipoamide in the process. The lipoamide dehydrogenase component, subunit 1:3 t EC I .S. 1.4), transfers the hydrogen from dihydrolipoamide via its I AD group to NAD, The glycine cleavage system (EC 1.4.4.2) and the dehydrogenases for pyruvate (EC1.2.4.1 (and alpha-ketogIutarate (EC2.3.1.6I) use the same enzyme subunit. The enzyme complex is inactivated by phosphorylation ([3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] kinase, EC2.7.1.115). and reactivated by dephosphorylation ([3-methyI-2-axobutanoate dehydrogenase (lipoamide)}-phosphatase. EC?.1.3.52). Branched-chain alpha-keto acid dehydrogenase is defective in maple syrup disease, which affects the breakdown of all three branched-chain amino acids.

The acyl-CoA dehydrogenase responsible for oxidation of alpha-methy Ibutyryl-CoA to tiglyJ-CoA (Andresen el al.. 2000) is short-branched-chain acyl-CoA dehydrogenase (ECl.3.99.2, FAD-containing). Short-chain acyl-CoA dehydrogenase (ECI.3.99.2), and possibly additional acyl-CoA dehydrogenases, also appear to oxidize alphamet hylbutyryl-Co A, though with tower specific activ ity.

An iron-sulfur flavoenzyme associated with the acyl-CoA dehydrogenases, ETF dehydrogenase (EC 1.5.5.1. FAD-containing), uses the reduced FAD-moiety of FTF (electron transfer flavoprotein I to generate the oxidative phosphorylation fuel ubiquinol.

The next three steps are catalyzed by enzymes otherwise used for short-chain fatty acid beta-oxidation. 3-hydroxybutyryl-CoA dehydratase (crotonase. EC4.2.I.55). beta-hydroxyacyl-CoA dehydrogenase (ECI. 1.1.35). and acetyl-CoA acy I transferase (EC2.3.I.I6), The intermediates generated by the last step. propionyf-CoA and acetyf-CoA, can then be metabolized further via typical reactions.

Storage

Body protein contains 4Kmg,g (Smith. 1980). Proteins of skeletal muscle are especially lie-rich (Mero, 1999). Protein breakdown during lasting, starvation, or severe illness provides much-needed amino acid precursors for the synthesis of acute-phase proteins.

Excretion

Healthy indiv iduals excrete very little lie. Whatever is shed or excreted into the intestines is reabsorbed quite efficiently via mechanisms described earlier. Most of the free lie filtered by the renal glomerulum is taken up from the proximal tubule via the sodium-driven brushbordei transporter B (Avissartvii/.. 2001). The rBAT (SLC3AI i-anchored heteroexchanger BATl'b" (Chairoungdua el al.. 1999) can augment the action of the bulk transporter. Transport across the basolateral membrane depends mainly on the 4F2 (SLC3A2) glycoprotein-anchored transporter LAT2 (SLC7AX).

coon

Hjft-CH

Vatno flmelftyl 2<piövaH.fiiiB

{PLP1

u-keto I-valine iscvalerale

BranGhsd-ehaki r ■ i *> iK:Kl lonsjni.nase jPLPj

L-giulamaro i.-keio-gMaist«

COOH

Ji-ketD (i-methytvalerBls CoA-SH MAD ^ Branchoü-c^n a -koto aCM! dehydrogenase (TPP iipöftmösi

NACH

HJC/' \h, CH, iiMotiiytuityryl-CoA

f rty

Snofttvancfled. chain «cyi-CijA [ jf dehydrogenase t (f*o.k. ,a IFAO) j BTF PADH; SCo*

TpglylCoA

S-CoA

H-HyUroiyacylCoA dehydrogenase

^NAD

SOjA

,. Moiriviacetoacölyi-CoA CoASH

Acalyl-CoA acyl transferase

NADH

PropmnyM^iA - Acotyl-CoA

Suconyl CoA

Figure 8.63 Metabolism of L isoleucme

Regulation

Branchcd-chain alpha-keto acid dehydrogenase (EC2.6.I.42) is the key regulatory enzyme for He (and L-valine'L-leueine) metabolism, which helps preserve this essential amino acid for protein synthesis during starvation. I he enzyme is inactivated by a specific, calmodulin-dependent kinase ([3-methyI-2-oxobu ta noate dehydrogenase {lipoamidet] kinase: EC2.7.I.1I5) and activated by a specific phosphatase ([3-methy l-2-oxobut a noate dehydrogenase (lipoamide)]-phosphatase; EC3.1.3.52). Branchcd-chain alpha-kcto acid dehydrogenase is also inhibited by the products of the reaction it catalyzes. isovaleryl-CoA. isobutyryl-CoA. and 2-niethyIbutyryI-CoA.

An intake imbalance with inadequacy of one of the BCAAs (Vat. leucine, or isoleucine) causes activation of orbitofronlal cortex, frontal gyrus, and thalamus and leads to the development of conditioned taste aversion (Gietzen and Magrum, 2001). Expression of several genes in response to inadequacy changes, bin the mechanisms responsible for learning to balance branchcd-chain amino acid intake remain elusive.

Function

Energy fuel: Eventually, most lie is broken down, providing about 5» kcal/g. As for L-leucine and l,-\aline, the main site of lie oxidation is skeletal muscle. Liver, intestines and other organs are responsible for much smaller percentages of lie utilization as an energy fuel.

Protein synthesis: Most human proteins contain lie as part of their specific sequence. A lack of this essential amino acid would limit their synthesis, therefore.

References

Andresen BS, Christensen E, Corydon TJ, Bross I1, Pilgaard B. Wanders RJ. Ruiter JP. Simonscn I I, Winter V Knudsen I, Schroeder LD, Gregersen N. Skovby P. Isolated 2-methyIbutyrylgtycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucineand valine metabolism. Am J Hum Genet 2000;67:1095-103 Avissar NE, Ryan CK, Ganapathy V Sax HC. Na^-dependent neutral amino acid transporter AT Bo is a rabbit epithelial cell brush border protein. Am J Physiol Cell Physiol 2001;28l:C963-71

Chairoungdua A. Segawa H, Kim JY. Miyamoto K. Hay a H, Fukui V. Mizoguchi K. ho H, Takeda I , Lndou II, Kauai Y. Identification of an amino acid transporter associated with thecystinuria-rclated type II membrane glycoprotein.,/BiolChern 1999;274:28845-8 Dancis J, llutzler J. lada K. Wada Y, MorikawaT, Arakawa T. Hypervafinemia: a defect in valine transamination. Pediatrics 1967:39:813 17 Gielzen DW, Magrum LJ. Molecular mechanisms in the brain involved in the anorexia of branchcd-chain amino acid deficiency, JNutr 2001:85 IS S55S Harris RA, HawesJW, Popov KM, Zhao Y, ShimomuraY, Sato J, JaskiewiezJ, Hurley TD. Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases. Adv Enzyme Reg 1997:37:271-93

JanssonT. Amino acid transporters in the human placenta. Fediatr Res 2IH) 1,44): 141 7 Mero A. Leucine supplementation and intensive training. Spans Med 1999:27:347- 58 Ritchie JW. Taylor I'M. Role of the System L permease LATI in amino acid and iodothy-

ronine transport in placenta. Btochem J 2001:356:719 25 Smith Kl I Comparative amino acid requirements, hut Sutr Soc 1980:39:71 S Young VR. Uorgonha S. Nitrogen and amino acid requirements; the Massachusetts Institute o ("Technology amino acid requirement pattern. J Nutr 2000:130:1841S-49S

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