Branched-Chain Amino Acids: Metabolism, Physiological

Function, and Application

Branched-Chain Amino Acids: Enzyme and Substrate Regulation1–3

John T. Brosnan4 and Margaret E. Brosnan
Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada

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ABSTRACT The three branched-chain amino acids (BCAAs) are the most hydrophobic of the amino acids and play
crucial roles in determining the structures of globular proteins as well as the interaction of the transmembrane
domains of membranous proteins with phospholipid bilayers. However, the three BCAAs do not behave identically. In
terms of protein secondary structure, valine and isoleucine exhibit a definite preference for the b-structure, whereas
leucine has a higher preference for the a-helix. Although mutation of one BCAA to another is commonly regarded as
conservative, there are well-documented examples of such substitutions that have a significant effect on protein
function. The occurrence of BCAA in nature is, therefore, attributable to their primary role in protein structure, not to
their secondary metabolic roles. These functions are important for almost all proteins; therefore, BCAA commonly
account for ;20–25% of most dietary proteins. Dietary BCAA largely escape first-pass splanchnic metabolism. The
first steps in their catabolism are common to all three, involving the BCAA aminotransferase (BCAT) and branched-
chain a-keto acid dehydrogenase (BCKD). Their further metabolism employs distinct pathways to different end-
products (glucose and/or ketone bodies). However, the fact that the flux-generating step for the catabolism of the
three BCAAs occurs at one of the common steps indicates that the production of these downstream products are not
individually regulated and, hence, may not play important individual roles. The catabolism of the BCAAs is highly
regulated by both allosteric and covalent mechanisms. BCKD is inhibited by phosphorylation and activated by
dephosphorylation. Allosteric inhibition of the kinase by the branched-chain keto acids (BCKA) (particularly by
a-ketoisocaproate) serves both as a mechanism for promoting the catabolism of excess quantities of these amino
acids as well as for conserving low concentrations of these dietary essential amino acids. Cytosolic and mitochondrial
isoenzymes of BCAT have been identified. They are thought to play an important role in brain neurotransmitter
metabolism. J. Nutr. 136: 207S–211S, 2006.

KEY WORDS:  leucine  isoleucine  valine  protein structure  muscle metabolism

The metabolism of BCAAs presents a number of novel
features: the catabolism of these three amino acids is controlled
by a common flux-generating step, their catabolic disposal
occurs largely in skeletal muscle, their circulating concen-
trations can influence the brain uptake of precursor amino acids
for neurotransmitter synthesis, and they can regulate protein
synthesis in a variety of tissues. Despite these unusual, or even
unique, features it is quite certain that the most important
function of these amino acids lies in their roles in proteins. In
this article we discuss the role of the BCAAs in protein
1
Published in a supplement to The Journal of Nutrition. Presented at the
conference ‘‘Symposium on Branched-Chain Amino Acids,’’ held May 23–24, 2005
in Versailles, France. The conference was sponsored by Ajinomoto USA, Inc. The
organizing committee for the symposium and guest editors for the supplement
were Luc Cynober, Robert A. Harris, Dennis M. Bier, John O. Holloszy, Sidney M.
Morris, Jr., and Yoshiharu Shimomura. Guest editor disclosure: L. Cynober, R. A.
Harris, D. M. Bier, J.O. Holloszy, S. M. Morris, Y. Shimomura: expenses for travel
to BCAA meeting paid by Ajinomoto USA; D. M. Bier: consults for Ajinomoto USA;
S. M. Morris: received compensation from Ajinomoto USA for organizing BCAA
conference.
2
Author Disclosure: J. Brosnan consults for Ajinomoto USA.
3
Supported by grants from the Canadian Institutes of Health Research.
4
To whom correspondence should be addressed. E-mail: jbrosnan@mun.ca.
structure, their metabolic disposal, the key enzymes of their
metabolism, and the regulation of these enzymes.
BCAAs AND PROTEIN STRUCTURE
Why BCAAs? Leucine, isoleucine, and valine are among
the most hydrophobic of amino acids. In fact, by some measures
of hydrophobicity they are the most hydrophobic (1). This is
a crucial determinant of their role in globular proteins, mem-
branous proteins, and coiled-coil structures. Generally, the
interior of water-soluble globular proteins consists, largely, of
hydrophobic amino acids, principally leucine, isoleucine, valine,
phenylalanine, and methionine. This is important, not only for
the stability of the folded protein, but also for the folding
pathway that leads to the mature structure (2). It is also im-
portant for the function of some globular proteins; for example,
the hydrophobic residues create a nonaqueous environment
that is important for oxygen binding in myoglobin and
hemoglobin, and for substrate binding and catalysis in a variety
of enzymes (3). A few globular proteins are not water soluble,
but lipid soluble. For example, lung surfactant protein B must

0022-3166/06 $8.00 Ó 2006 American Society for Nutrition.

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208S SUPPLEMENT
interact with phospholipids in surfactant, so it is among the
most hydrophobic of proteins. BCAAs make up 37% of the
amino acid composition of surfactant protein B (17.7% leucine,
11.4% valine, 7.6% isoleucine) (4). The dominant structural
feature of this protein is its amphipathic helices, with the
branched-chain residues interacting with lipid acyl chains, and
positively charged groups interacting with the lipid head groups
(4). Membranous proteins require hydrophobic amino acids in
their transmembrane domains for interaction with the hy-
drocarbon chains of fatty acids. For example, glycophorin, a
protein in the red cell membrane, contains a total of 11 BCAAs
in the 19 residues of the a-helix that crosses the membrane (5).
Another important role of leucine and its hydrophobic
partners occurs in coiled-coiled a-helices in such proteins as
myosin, fibrinogen, keratin, and a number of transcription fac-
tors. Each polypeptide of the rod-like coiled coil has an amino
acid sequence containing a number of heptad repeats, with
leucine often in the fourth position, and another hydrophobic
residue in the first position. When the helices twist, there are
3.5 residues per turn of the helix and the helices are packed
together by hydrophobic interactions between the leucyl
residues and the residues in the 1 position (6). In transcription
factors, coiled coils, referred to as leucine zippers, permit
formation of homodimers and/or heterodimers, which are the
functionally active form of such transcription factors as Fos and
Jun (7).
Although a variety of weak interactions (hydrogen bonds,
salt-linkages, van der Waals interactions, hydrophobic inter-
actions) are key determinants of protein structure, their relative
importance is not invariant. The proteins of thermophilic organ-
isms, in particular, their relative resistance to unfolding at high
temperatures, have been a source of fascination for protein
chemists. Undoubtedly, many factors play a role in this thermo-
stability: among them, it has been suggested that hydrophobic
interactions may be particularly important as the strength of
these interactions increases with increasing temperature (8). A
comparison of the amino acid composition of 110 pairs of
homologous proteins from mesophilic and thermophilic organ-
isms showed that the thermophilic proteins have both higher
hydropathy and charged amino acid composition; the increase
in hydropathy was largely accounted for by their higher leucine
composition (9).
Why three BCAAs? Despite their similarity, the three
BCAAs play subtly different roles in proteins. Their side-
chains differ in size, shape, and hydrophobicity. Therefore,
they have different predilections for different secondary
structure motifs. Leucine is more common in a-helices than
in b-sheets, whereas the reverse is true for valine and
isoleucine (10). These preferences account for the key role
of leucine in helical zipper structures. The differences also
account for the fact that these amino acids are not always
interchangeable in proteins. There are, of course, many
occasions when such substitutions are conservative but this
is not invariable. For example, a substitution of isoleucine for
valine (V122I) in transthyretin results in a cardiomyopathy
characterized by amyloid deposition in the heart (11). A valine
for leucine substitution (L162V) in the peroxisome prolifer-
ator-activated receptor-a results in an altered plasma lipid
profile (12) whereas a leucine for valine substitution (V836L) in
the extracellular calcium receptor of the TM6/TM7 protein leads
to autosomal dominant hypocalciuria (13).
All three of the BCAAs are readily produced under con-
ditions designed to mimic prebiotic organic synthesis (14).
They have been retained because of their critical roles in pro-
tein structure. Most proteins have a relatively high proportion
of these amino acids. Indeed, most dietary proteins consist of
;20% BCAAs. They comprise some 35% of the indispensible
amino acid requirements of mammals (15).
METABOLIC DISPOSAL OF THE BCAAS
Unusual aspects of BCAA metabolism. BCAA metabo-
lism is characterized by a number of unusual features. First,
although these amino acids play a number of regulatory roles
(e.g., in muscle protein synthesis, insulin secretion, and brain
amino acid uptake), they are not metabolized to unique bio-
logically active molecules. Second, their catabolism involves two
common steps and, indeed, the flux-generating step, branched-
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chain keto acid dehydrogenase (BCKD)5, is a common step (Fig.
1)(16); therefore, BCAAs tend to be catabolized in lockstep.
Third, dietary BCAAs largely escape first-pass hepatic catabolism.
Tissue distribution of BCAA catabolism. The capacity of
different tissues to catabolize BCAAs in humans is shown in
Figure 2. This capacity was calculated by multiplying the
activities of the branched-chain amino acid aminotransferase
(BCAT) and the BCKD in different tissues (17) by the mass of
these tissues in a 70 kg human (18). In the case of the BCKD,
only the active component was computed. The results are
striking. It is clear that one of the distinguishing features of
BCAA catabolism is the relatively small fraction of the capacity
that resides in the liver. About half of the capacity resides in
skeletal muscle, whereas a considerable portion of the activity
also resides in adipose tissue. These results are only suggestive
because other factors such as rates of blood flow, and transport
into cells, are also important. Nevertheless, they are consistent
with a body of experimental evidence. For example, Wahren
et al. (19) showed that after ingestion of a protein-rich meal the
three BCAAs accounted for .50% of the splanchnic output of
amino acids even though they only comprise 20% of the protein
source (beef) that was ingested. Clearly, they largely escaped
splanchnic catabolism. Elia and Livesey (20) examined human
forearm metabolism after ingestion of a steak. They observed
a marked increased in BCAA uptake into muscle, but only
a minor increase in BCKA output. A similar observation was
made after leucine infusion. Clearly, muscle is a major site for
BCAA catabolism.
Why should the BCAAs be catabolized extra-hepatically?
Liver contains the urea cycle as well as the catabolic enzymes
for most of the amino acids. However, it is now apparent that
the textbook statement that liver oxidizes most of the dietary
amino acids cannot be true. The oxidation of 100 g of dietary
protein obliges the consumption of ;3.8 mol of O2 per d.
However, the liver only consumes ;3 mol of O2 per d (20), so
that even if no other dietary substrate were consumed by the
liver (i.e., no carbohydrate, fat, or alcohol) it would be im-
possible to completely oxidize dietary amino acids to CO2 and
H2O. It is now apparent that this problem is resolved in two
ways. First, the liver does not completely oxidize many amino
acids but converts them to glucose and acetoacetate, even in
the fed state (21). Second, a considerable portion of dietary
amino acids are metabolized extra-hepatically, including the
appreciable first-pass intestinal metabolism of many amino
acids (22) and the muscle catabolism of the BCAAs.
Consequences and implications of the common steps in
BCAA catabolism. The second unusual feature of BCAA
metabolism is that the first two enzymes of their catabolic
5
Abbreviations used: BCAT, branched-chain amino acid aminotransferase;
BCKA, branched-chain keto acid; BCKD, branched-chain keto acid dehydroge-
nase.
 BRANCHED-CHAIN AMINO ACID METABOLISM
disposal, the aminotransferase and the flux-generating de-
hydrogenase, are common to the three amino acids (Fig. 1).
This, no doubt, accounts for the remarkable correlation among
the plasma levels of the three amino acids in a variety of
situations, such as in Type 1 (23) and Type 2 diabetes (24)
(Fig. 3). These data suggest that the plasma concentrations of
the individual BCAAs are not tightly defended and therefore
not particularly important. Nor does the catabolic fate of the
different BCAAs appear to be an important consideration.
Leucine is ketogenic, valine is glucogenic, and isoleucine is
both glucogenic and ketogenic. However, the fact that the
catabolism of all three BCAAs is regulated at a common step
suggests that it is not primarily driven by a need for glucose or
a need for ketone bodies.
FIGURE 2 Human BCAA catabolic capacity. Tissue distribution of
BCAT (1) and BCKD (2) in human tissues. The total BCAT activity in a 70
kg human was calculated to be 6784 U; the total BCKD activity was
calculated to be 77 U.
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FIGURE 1 The BCAA catabolic pathway.
TCA, tricarboxylic acid cycle; KIV, a-ketoisoval-
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erate; KMV, a-keto-b-methylvalerate; CoA-SH,
reduced coenzyme A; IB-CoA, isobutyryl-CoA;
MB-CoA, a-methylbutyryl-CoA; IV-CoA, isova-
leryl-CoA; R-CoA, acyl-CoA. Reproduced with
permission from Shimomura et al. (16).
Interorgan aspects of BCAA metabolism. Muscle is not a
gluconeogenic tissue. Therefore, if valine and isoleucine are
to be converted to glucose they cannot be completely oxidized
in this tissue. Our own work with [1-14C]- and [U-14C]-
a- ketoisocaproate has shown that this keto acid is almost
completely oxidized in the perfused rat heart (25). However,
similar experiments showed that a-ketoisovalerate is not; we
identified b-hydroxyisobutyrate (an intermediate in the valine
catabolic pathway) as a major end product of a-ketoisovalerate
catabolism. Lee and Davis (26) obtained similar results in the
isolated perfused hind-limb. Plasma levels of b-hydroxyisobu-
tyrate are reported to be 21 mM and 97 mM, respectively, in
control and 3-d fasted humans (27). A special feature of the
valine catabolic pathway is that CoA is hydrolyzed off one of
the intermediates (b-hydroxyisobutyryl CoA) and subsequently,
reintroduced at the level of methylmalonic semialdehyde.
Shimomura et al. (28) view this as a means of reducing the
concentration of methylacrylyl CoA, which is a highly reactive
intermediate. Additionally, b-hydroxyisobutyrate can serve as
an inter-organ gluconeogenic substrate. We have shown that
b-hydroxyisobutyrate is a good glucogenic substrate in both
hepatocytes and renal cortical tubules (29). These studies
extend the classic work of Chang and Goldberg (30), with iso-
lated diaphragms, that show isoleucine and valine as major
carbon sources for glutamine synthesis.
ENZYME EXPRESSION AND REGULATION
Branched-chain amino acid aminotransferase. Work from
Hutson’s laboratory has shown that there are two BCAT
isoenzymes, one located in mitochondria and the other in the
cytosol (17). These are different gene products. In rats, the
mitochondrial isoenzyme is expressed ubiquitously, whereas the
cytosolic activity is restricted to brain, ovary, and placenta (31).
210S
FIGURE 3 Relations among the BCAAs in ZDF fa/fa rats (at 5 and
11 wk of age) and their lean controls. Reproduced with permission from
Wijekoon et al. (24).
In humans, the mitochondrial activity is widely distributed,
with particularly high expression in the colon, kidney, and
skeletal muscle and a very low expression in the liver (17). Of
human tissues examined, cytoplasmic activity was found to be
expressed only in the brain; however, ovarian and placental
tissues were not examined (17). A marked induction of the
mitochondrial BCAT in mammary epithelial cells has been
found during late pregnancy and lactation in the rat (32).
The mitochondrial BCAT isoenzyme is primarily responsible
for initiating BCAA catabolism. Hutson et al. (33) have made
a novel proposal for the function of the cytosolic isoenzyme in
brain. A BCAA-based nitrogen shuttle is hypothesized that
would play an important role in glutamergic neurons (Fig. 4).
Glutamate is the major excitatory neurotransmitter in the
central nervous tissue. Upon release into synapses, it initiates
neurotransmission and is then efficiently taken up by astroglial
cells, which convert it to glutamine, via glutamine synthetase.
SUPPLEMENT
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FIGURE 4 BCAA shuttle and brain glutamate metabolism. Gln’ase,
glutaminase; GDH, glutamate dehydrogenase; aKg, a-ketoglutarate;
OAA, oxaloacetate; PC, pyruvate carboxylase; TCA, tricarboxylic acid.
Reproduced with permission from Hutson et al. (33).
This glutamine is released to neurons that employ glutaminase
to convert it back to glutamate, thus completing the cycle.
However, this cycle is not 100% efficient, and some of
the glutamate is oxidized in the astroglia and must be replaced,
via anaplerotic mechanisms involving pyruvate carboxylase.
a-Ketoglutarate, produced in astroglial mitochondria in this
process, is transaminated to glutamate by the mitochondrial
BCAT. The branched-chain keto acids (BCKAs) produced are
thought to be transferred to the neurons where they can be
transaminated back to BCAA by the cytoplasmic BCAT. The
ultimate source of neuronal amino groups is ammonia, which is
fixed into glutamate by glutamate dehydrogenase.
Two pieces of evidence support such a shuttle. First, in the
central nervous system, the mitochondrial BCAT is neuronal,
whereas the cytosolic isoenzyme is found in the astroglia.
Second, gabapentin, an inhibitor of the cytoplasmic BCAT,
inhibits the transamination of leucine and the de novo synthe-
sis of glutamate and glutamine in rat retinal preparations (17).
Hutson’s group has extended its studies of these enzymes to
a variety of organs and to peripheral nerves (34). The important
findings are that the mitochondrial BCAT co-localized with
BCKD (very high expression of both enzymes were found in
various secretory cells), whereas the cytosolic BCAT occurred
in cells that do not express BCKD. This localization of the cy-
tosolic isoenzyme certainly points to a physiological role other
than in BCAA oxidation.
Branched-chain keto acid dehydrogenase. BCKD is the
flux-generating step for BCAA catabolism. Classic isotopic
work in humans, by Matthews et al. (35), showed that leucine
transamination was much faster than the decarboxylation of
a-ketoisocaproate. This irreversible oxidative decarboxylation
is catalyzed by an enzyme complex, the BCKD complex, which
consists of multiple copies of the BCKA decarboxylase (E1),
dihydrolipoamide acyltransferase (E2), and dihydrolipoamide
dehydrogenase (E3). A genetic defect in the BCKD complex is
responsible for maple syrup urine disease. The activity of the
BCKD is regulated by both covalent and allosteric mechanisms.
Phosphorylation of E1 (on its a subunit) by the BCKD kinase
inhibits its activity; this may be reversed by dephosphorylation
via a phosphatase. The BCKD kinase has been isolated,
characterized, and cloned (36) and is considered to be the key
regulator of BCKD activity, although this perception may need
to be modified when more is known about the phosphatase.
The kinase is regulated in two different ways. It is allosterically
 BRANCHED-CHAIN AMINO ACID METABOLISM
inhibited by a-ketoisocaproate (and by the other BCKAs,
although they are less effective), which provides an elegant
means of enhancing BCAA disposal when they are present in
excess and of conserving these essential amino acids when they
are less available. This effect of a-ketoisocaproate also explains
the finding that the provision of excess leucine results in
decreased circulatory levels of valine and isoleucine. The
BCKD kinase also appears to be regulated by its association
with the BCKD complex, although the molecular details of this
interaction have not yet been established. Shimomura et al.
(16) have reviewed the regulation of BCKD kinase expression
by nutritional, hormonal, and pathological factors. The active
dephosphorylated BCKD is also susceptible to allosteric
inhibition, in particular by NADH and by the CoA esters
that arise during BCAA catabolism.
PERSPECTIVE
How well do we understand BCAA metabolism? We have
learned a great deal about BCAA metabolism, both in vivo and
in vitro, in recent years. Although it is regarded as a mature
field of study, we may wonder at the extent of our knowledge.
The occurrence of the diseases hypervalinemia (37) and
hyperleucinemia-hyperisoleucinemia are, apparently, due to
the impaired transamination of valine alone, or of both leucine
and isoleucine. Such disorders challenge the conventional view
that each of the BCAAs is transaminated by a common
aminotransferase. It seems likely that these diseases are due to
mutations in the common aminotransferase and that they affect
its ability to act on each of the BCAAs. Nevertheless, this has not
been established.
Is our knowledge of BCAA metabolism sufficient to con-
struct a mathematical model and, if so, how well will it accom-
modate experimental observations? Our knowledge of the
kinetic properties of the components of BCAA catabolism is
probably sufficient to permit the construction of a robust model.
After decades of biochemical reductionism we should aim to
reconstruct the system. Such reconstructions can be very re-
vealing. For example, modeling of a linear pathway of yeast
glycolysis, with all the known in vitro kinetic properties of the
constituent enzymes, failed to predict a stable steady-state.
However, a closer match of the model to the experiment could
be attained by the inclusion of branches, such as toward
glycogen, trehalose, etc. (38). In this era of Systems Biology, it
behooves us to subject our knowledge of BCAA metabolism to
a comparable test.
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