Proteins and Amino Acids1
Dwight E. Matthews
1Abbreviations: ATP, adenosine triphosphate; AV; arteriovenous; BCAA, branched-chain amino acid; CO2, carbon dioxide; CoA, coenzyme A; DAAO, direct amino acid oxidation; EAR, estimated average requirement; FAO/WHO/UNU, Food and Agriculture Organization/World Health Organization/United Nations University; IAAO, indicator amino acid oxidation; IDAA, indispensable amino acid; KIC, α-ketoisocaproate; N, nitrogen; NH3, ammonia; PER, protein efficiency ratio; RDA, recommended dietary allowance; TCA, tricarboxylic acid; TML, trimethyllysine.
Proteins are associated with all forms of life, and much of the effort to determine how life began has centered on how proteins were first produced. Amino acids joined together in long strings by peptide bonds form proteins that twist and fold in three-dimensional space and produce centers to facilitate the biochemical reactions of life that either would run out of control or not run at all without them. Life could not have begun without these enzymes, thousands of different types of which are found in the body. Proteins are prepared and secreted to act as cell-cell signals in the form of hormones and cytokines. Plasma proteins produced and secreted by the liver stabilize the blood by forming a solution of the appropriate viscosity and osmolarity. These secreted proteins also transport a variety of compounds through the blood.
The largest source of protein in higher animals resides in muscle. Through complex interactions, entire sheets of proteins slide back and forth to form the basis of muscle contraction and all aspects of our mobility. Muscle contraction provides for pumping oxygen and nutrients throughout the body, for inhalation and exhalation of our lungs, and for movement. Many of the underlying causes of noninfectious diseases are the result of derangements in the sequence of proteins. The incredible advances in molecular biology provided tremendous information about DNA and RNA and introduced the field of genomics. This research is not driven to understand DNA itself, but rather to understand the purpose and function of the proteins that are translated from the genetic code. The emerging field of proteomics studies the expression, modification, and regulation of proteins.
Three major classes of substrates are used for energy: carbohydrates, fat, and protein. Protein differs from the other two primary sources of dietary energy by inclusion of nitrogen (N). Protein on average is 16% by weight N. The component amino acids of proteins contain one N in the form of an amino group and additional N, depending on the amino acid. When amino acids are oxidized to carbon dioxide (CO2) and water to produce energy, N is also produced as a waste product that must be eliminated via incorporation into urea. Conversely, N must be available when the body synthesizes amino acids de novo. The synthetic routes of other N-containing compounds in the body (e.g., nucleic acids for DNA and RNA synthesis) obtain their N during synthesis from donation of N from amino acids. Therefore, when we think of amino acid metabolism in the body, we really mean N metabolism.
Protein and amino acids are also important to the energy metabolism of the body. As Cahill pointed out (1),
protein is the second largest store of energy in the body after adipose tissue fat stores (Table 1.1). Carbohydrate is stored as glycogen, and although it is important for short-term energy needs, it is of very limited capacity for providing for energy needs beyond a few hours. Amino acids from protein are converted to glucose by the process called gluconeogenesis to provide a continuing supply of glucose after the glycogen is consumed during fasting. Conversely, however, protein stores must be conserved for the numerous critical roles in which protein functions in the body. Loss of more than approximately 30% of body protein results in reductions in muscle strength for breathing, immune function, organ function and, ultimately, in death. Hence, the body must adapt to fasting by conserving protein, as is seen by a dramatic decrease in N excretion within the first week of onset of starvation.
protein is the second largest store of energy in the body after adipose tissue fat stores (Table 1.1). Carbohydrate is stored as glycogen, and although it is important for short-term energy needs, it is of very limited capacity for providing for energy needs beyond a few hours. Amino acids from protein are converted to glucose by the process called gluconeogenesis to provide a continuing supply of glucose after the glycogen is consumed during fasting. Conversely, however, protein stores must be conserved for the numerous critical roles in which protein functions in the body. Loss of more than approximately 30% of body protein results in reductions in muscle strength for breathing, immune function, organ function and, ultimately, in death. Hence, the body must adapt to fasting by conserving protein, as is seen by a dramatic decrease in N excretion within the first week of onset of starvation.
TABLE 1.1 BODY COMPOSITION OF A NORMAL MAN IN TERMS OF ENERGY COMPONENTS | ||||||||||||||||||||||||||||||||
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Body protein is made up of 20 different amino acids, each with different metabolic fates in the body, with diverse activities in different metabolic pathways in different organs, and with varying compositions in different proteins. When amino acids are liberated after absorption of dietary protein, the body makes a complex series of decisions concerning the fate of those amino acids: to oxidize them for energy, to incorporate them into proteins in the body, or to use them in the formation of a number of other N-containing compounds. The purpose of this chapter is to elucidate the complex pathways and roles amino acids play in the body, with a focus on nutrition.
AMINO ACIDS
Basic Definitions
The amino acids that we are familiar with and all those incorporated into mammalian protein are “α”-amino acids. By definition, they have a carboxyl-carbon group and an amino N group attached to a central α-carbon (Fig. 1.1). Amino acids differ in structure by the substitution of one of the two hydrogens on the α-carbon with another functional group. Amino acids can be characterized by their functional groups, which are often organized at neutral pH into the classes of (a) nonpolar, (b) uncharged but polar, (c) acidic (negatively charged), and (d) basic (positively charged) groups. Within any class are considerable differences in shape and physical properties. Thus, amino acids are often grouped into other functional subgroups. For example, amino acids with an aromatic group—phenylalanine, tyrosine, tryptophan, and histidine—are often associated together, although tyrosine is clearly polar and histidine is also basic. Other common groupings are the aliphatic or neutral amino acids (glycine, alanine, isoleucine, leucine, valine, serine, threonine, and proline). Proline is different in that its functional group is also attached to the amino group, thus forming a fivemembered ring. Because of the ring, proline is actually an imino acid, not an amino acid. Serine and threonine contain hydroxyl groups. There is also another important subgroup: the branched-chain amino acids (BCAAs: isoleucine, leucine, and valine), which share common enzymes for the first two steps of their degradation. The acidic amino acids, aspartic acid and glutamic acid, are often referred to as their ionized, salt forms: aspartate and glutamate. These amino acids become asparagine and glutamine when an amino group is added in the form of an amide group to their carboxyl tails.
The sulfur-containing amino acids are methionine and cysteine. Cysteine is often found in the body as an amino acid dimer called cystine in which the thiol groups (the two sulfur atoms) are connected to form a disulfide bond. Particular attention should be paid when reading the literature to note the distinction between the names cysteine and cystine, because the former is a single amino acid, and the latter is a dimer with different properties. Other amino acids that contain sulfur, such as homocysteine, are not incorporated into protein.
All amino acids exist as charged particles in solution: in water, the carboxyl group rapidly loses a hydrogen to form a carboxyl anion (negatively charged), whereas the amino group gains a hydrogen to become positively charged. The amino acids therefore become “bipolar” (often called a zwitterion) in solution, but without a net charge (the positive and negatively charges cancel). The attached functional group may distort that balance, however. The acidic amino acids lose the hydrogen on the second carboxyl group and become negatively charged in solution. In contrast, the basic group amino acids in part accept a hydrogen on the second N and form a molecule with a net positive charge. Although the other amino acids do not specifically accept or donate additional hydrogens in neutral solution, their functional groups do influence the relative polarity and acid-base nature of the bipolar portion of the amino acids and give each amino acid different properties in solution.
The functional groups of amino acids also vary by size. The molecular weights of the amino acids are shown in Table 1.2. Amino acids range from the smallest, glycine, to the large and bulky molecules (e.g., tryptophan). Most amino acids crystallize as uncharged molecules when they
are purified and dried. The molecular weights shown in Table 1.2 reflect their molecular weight as crystalline amino acids. The basic and acidic amino acids tend to form much more stable crystals as salts, however, rather than as free amino acids. Glutamic acid can be obtained as the free amino acid with a molecular weight of 147 and as its sodium salt, monosodium glutamate, which has a crystalline weight of 169. Lysine is typically found as a salt containing hydrogen chloride. Therefore, when amino acids are represented by weight, it is important to know whether the weight is based on the free amino acid or on its salt.
are purified and dried. The molecular weights shown in Table 1.2 reflect their molecular weight as crystalline amino acids. The basic and acidic amino acids tend to form much more stable crystals as salts, however, rather than as free amino acids. Glutamic acid can be obtained as the free amino acid with a molecular weight of 147 and as its sodium salt, monosodium glutamate, which has a crystalline weight of 169. Lysine is typically found as a salt containing hydrogen chloride. Therefore, when amino acids are represented by weight, it is important to know whether the weight is based on the free amino acid or on its salt.
 Fig. 1.1. Structural formulas of the 21 common α-amino acids. The α-amino acids all have a carboxyl group, an amino group, and a differentiating functional group attached to the α-carbon. The generic structure of amino acids is shown in the upper left corner with the differentiating functional group marked by R. The functional group for each amino acid is shown below. Amino acids have been grouped by functional class. Proline is actually an imino acid because of its cyclic structure involving its nitrogen (N). |
Another important property of amino acids is optical activity. Except for glycine, which has its functional group as a single hydrogen, all amino acids have at least one chiral center: the α-carbon. The term “chiral” comes from Greek for hand in that these molecules have a left (“levo” or “L”) and right (“dextro” or “D”) handedness to them around the α-carbon atom. Because of the tetrahedral structure of the carbon bonds, there are two possible arrangements of a carbon center with the same four different groups bonded to it that are not superimposable; the two configurations, called stereoisomers, are mirror images of each other. The body recognizes only the L-form of amino acids for most reactions in the body, although some enzymatic reactions will operate with a lower efficiency when given the D-form. Because we do encounter some D-form amino acids in the foods that we eat, the body has some mechanisms for clearing these amino acids through renal filtration.
TABLE 1.2 COMMON AMINO ACIDS IN THE BODY | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Any number of molecules could be designed that complete the basic definition of an amino acid: a molecule with a central carbon to which an amino group, a carboxyl group, and a functional group are attached. A relatively limited variety appears in nature, however, and only 20 are incorporated directly into mammalian protein. Amino acids are selected for protein synthesis when coupled to transfer RNA (tRNA). To synthesize protein, strands of DNA are transcribed into messenger RNA (mRNA). tRNA binds to mRNA in 3-base groups. Different combinations of 3 consecutive RNA molecules in the mRNA code for different tRNA molecules. However, the 3-base combinations of mRNA are recognized by only 20 different tRNA molecules, and 20 different amino acids are incorporated into protein during protein synthesis.
Of these 20 amino acids in proteins, some are synthesized de novo in the body either from other amino acids or from simpler precursors. These amino acids may be deleted from our diet without impairing health or blocking growth. These amino acids are nonessential and dispensable from the diet. No pathways exist for the synthesis of several other amino acids in humans, however, and hence these amino acids are essential or indispensable to the diet. The classification of amino acids as nondispensable/ dispensable or essential/nonessential for humans is shown in Table 1.2. Both the standard three-letter abbreviation and the one-letter abbreviation used in representing amino acid sequences in proteins are also presented in Table 1.2 for each amino acid. Some dispensable amino acids may become conditionally indispensable under conditions when synthesis becomes limited or when adequate amounts of precursors are unavailable to meet the needs of the body (2, 3, 4). The history and rationale of the classification of amino acids in Table 1.2 are discussed in greater detail later in this chapter.
Besides the 20 amino acids that are recognized by tRNA for incorporation into protein, other amino acids appear commonly in the body. These amino acids have important metabolic functions. Examples are ornithine and citrulline, which are linked to arginine through the urea cycle. Other amino acids appear as modifications of amino acids after they have been incorporated into proteins. Examples are hydroxyproline and hydroxylysine, which are produced when proline and lysine residues in collagen protein are
hydroxylated, and 3-methylhistidine, which is produced by posttranslational methylation of select histidine residues of actin and myosin proteins. Because no tRNA exists to code for these amino acids, they cannot be reused when a protein containing them is broken down (hydrolyzed) to its individual amino acids.
hydroxylated, and 3-methylhistidine, which is produced by posttranslational methylation of select histidine residues of actin and myosin proteins. Because no tRNA exists to code for these amino acids, they cannot be reused when a protein containing them is broken down (hydrolyzed) to its individual amino acids.
Amino Acid Pools and Distribution
The distribution of amino acids is complex. Not only are different amino acids incorporated into a variety of different proteins in many different organs in the body, but also amino acids are consumed in the diet from numerous protein sources. In addition, each amino acid is maintained in part as a free amino acid in solution in blood and inside cells. Overall, a wide range of concentrations is found among amino acids across the various protein and free pools that exist. We consume protein in food that is enzymatically hydrolyzed in the alimentary tract, thus releasing free individual amino acids that are then absorbed by the gut lumen and are transported into portal blood. Amino acids then pass into the systemic circulation and are extracted by different tissues. Although the concentrations of individual amino acids vary among different free pools such as plasma and intracellular muscle, the abundance of individual amino acids is relatively constant in a variety of proteins throughout the body and nature. Table 1.3 shows the composition of amino acids in hen egg protein, mammalian muscle and liver proteins (5), and human milk (6). The data are expressed as moles of amino acid. The historical expression of amino acids is on a weight basis (e.g., grams of amino acid). Comparing amino acids by weight skews the comparison toward the heaviest amino acids and makes them appear more abundant than they are. For example, tryptophan (molecular weight 204) appears almost three times as abundant as glycine (molecular weight 75) when quoted in terms of weight.
TABLE 1.3 AMINO ACID COMPOSITION OF SEVERAL DIFFERENT PROTEIN SOURCES | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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An even distribution of all 20 amino acids would be 5% per amino acid per protein, and the median amino acid content centers around this value for the proteins shown in Table 1.3. Tryptophan is the least common amino acid in many proteins. Considering the effect of tryptophan’s large size on the conformation of proteins, it is not surprising to find less tryptophan in protein. Other amino acids of modest size and limited polarity, such as alanine, leucine, serine, and valine, are relatively abundant in protein (8% to 10% per amino acid). Although the abundance of the indispensable amino acids (IDAAs) is similar across the protein sources in Table 1.3, various vegetable proteins are deficient or low in some IDAAs. In the body, certain proteins are particularly rich in specific amino acids to produce specific functions in the protein. For example, collagen is a fibrous protein abundant in connective tissues in tendons, bone, and muscle. Collagen fibrils are arranged in different ways, depending on the functional type of collagen. Glycine comprises approximately one third of collagen, and there is also considerable proline and hydroxyproline (proline converted after it has been incorporated into collagen). The glycine and proline residues allow the collagen protein chain to turn tightly and intertwine, and the hydroxyproline residues provide for hydrogen bond crosslinking. Generally, the alterations in amino acid concentrations do not vary dramatically among proteins as they do in collagen, but such examples demonstrate the diversity and functionality of the different amino acids in proteins.
The abundance of amino acids varies among amino acids over a far wider range in the free pools of extracellular and intracellular compartments. Typical values of free amino acid concentrations in plasma and in intracellular muscle are shown in Table 1.4. The primary points of Table 1.4 are as follows: (a) amino acid concentrations vary widely among amino acids, and (b) free amino acids are generally concentrated inside cells. Although the correlation between plasma and muscle free intracellular amino acid levels is significant, the relationship is not linear (7). Concentrations of plasma amino acids range from a low of approximately 20 µM for aspartic acid and methionine to a high of approximately 500 µM for glutamine. The median level for plasma amino acids is 100 µM. No defined relationship exists between the nature of amino acids (IDAAs versus dispensable amino acids) and amino acid concentrations or type of amino acids (e.g., plasma concentrations of the three BCAAs range from 50 to 250 µM). One notable point is that the concentrations
of the acidic amino acids, aspartate and glutamate, are very low outside cells in plasma. In contrast, the concentration of glutamate is among the highest inside cells, such as muscle (Table 1.4).
of the acidic amino acids, aspartate and glutamate, are very low outside cells in plasma. In contrast, the concentration of glutamate is among the highest inside cells, such as muscle (Table 1.4).
TABLE 1.4 TYPICAL CONCENTRATIONS OF FREE AMINO ACIDS IN THE BODY | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Important to bear in mind are the differences in the relative amounts of N contained in extracellular and intracellular amino acid pools and in protein itself. A physiologically normal person has approximately 55 mg of amino acid N/L outside cells in extracellular space and approximately 800 mg of amino acid N/L inside cells; this means that free amino acids are approximately 15-fold more abundant inside cells than outside cells (7). The second point is that the total pool of free amino acid N is small compared with protein-bound amino acids. Multiplying the free pools by estimates of extracellular water (0.2 L/kg) and intracellular water (0.4 L/kg) provides a measure of the total amount of N present in free amino acids: 0.33 g N/kg body weight. In contrast, body composition studies have shown that the N content of the body is 24 g N/kg body weight (8, 9). Therefore, free amino acids make up approximately 1% of the total amino N pool versus more than 99% of the amino acids that reside in proteins.
Amino Acid Transport
The gradient of amino acids within and outside cells is maintained by active transport. From a simple scan of Table 1.4, it is clear that different transport mechanisms must exist for different amino acids to produce the range of concentration gradients observed. Many different transporters exist for different types and groups of amino acids (10, 11, 12). Amino acid transport is probably one of the more difficult areas of amino acid metabolism to quantify and characterize. The affinities of the transporters and their mechanisms of transport set the intracellular levels of the amino acids. Generally, the IDAAs have lower intracellular/extracellular gradients than do the dispensable amino acids (Table 1.4) and are transported by different carriers. The amino acid transporters are membrane-bound proteins that recognize different amino acid shapes and chemical properties (e.g., neutral, basic, or anionic). Transport occurs both into and out of cells. Transport may be thought of as a process that sets the intracellular/extracellular gradient, or the transporters may be thought of as processes that set the rates of amino acid influx into and efflux from cells, which then define the intracellular/extracellular gradients (10). Perhaps the more dynamic concept of transport defining flows of amino acids is more appropriate, but in real life the gradient (e.g., intracellular muscle amino acid levels) is measurable, not the rates.
The transporters fall into two classes: sodiumindependent and sodium-dependent carriers. The sodiumdependent carriers cotransport a sodium atom into the cell along with the amino acid. The high extracellular/ intracellular sodium gradient (140 mEq outside and 10 mEq inside) facilitates the inward transport of amino acids by the sodium-dependent carriers. These transporters generally produce larger gradients and accumulations of amino acids inside cells than outside them. The sodium entering the cell may be transported out via the sodium-potassium pump that transports a potassium ion in for the removal of a sodium ion.
Few of the transporter proteins have been identified; most information concerning transport has been accrued through kinetic studies of membranes using amino acids and competitive inhibitors or amino acid analogs to define and characterize individual systems. Table 1.5 lists the different amino acid transporters characterized to date and the amino acids they transport. The neutral and bulky amino acids (the BCAAs, phenylalanine, methionine, and histidine) are transported by system L. System L is sodium independent and operates with a high rate of exchange and produces small gradients. Other important transporters are systems ASC and A. These transporters use the energy available from the sodium-ion gradient as a driving force to maintain a steep gradient for the various amino acids transported (e.g., glycine, alanine, threonine, serine, and proline) (10, 11). The anionic transporter (XAG-) also produces a steep gradient for the dicarboxylic amino acids, glutamate and aspartate. Other important carriers are system N and Nm for glutamine, asparagine, and histidine. System y+ handles much of the transport
of the basic amino acids. Some overall generalizations can be made in terms of the type of amino acid transported by a given carrier, but the system is not readily simplified because individual carrier systems transport several different amino acids, whereas individual amino acids are often transported by several different carriers with different efficiencies. Thus, amino acid gradients are formed and amino acids are transported into and out of cells via a complex system of overlapping carriers.
of the basic amino acids. Some overall generalizations can be made in terms of the type of amino acid transported by a given carrier, but the system is not readily simplified because individual carrier systems transport several different amino acids, whereas individual amino acids are often transported by several different carriers with different efficiencies. Thus, amino acid gradients are formed and amino acids are transported into and out of cells via a complex system of overlapping carriers.
TABLE 1.5 AMINO ACID TRANSPORTERS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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PATHWAYS OF AMINO ACID SYNTHESIS AND DEGRADATION
Several amino acids have their metabolic pathways linked to the metabolism of other amino acids. These codependencies that link the pathways of amino acids become important when nutrient intake is limited or when metabolic requirements are increased. Two aspects of metabolism are reviewed here: (a) synthesis of amino acids and (b) amino acid degradation. Degradation serves two useful purposes: (a) production of energy from the oxidation of individual amino acids (≈4 kcal/g protein, almost the same energy production as for carbohydrate) and (b) conversion of amino acids into other products. The latter is also related to amino acid synthesis: the degradation pathway of one amino acid may be the synthetic pathway of another amino acid. The other important aspect of amino acid degradation is production of other nonamino acid, N-containing compounds in the body. The need for synthesis of these compounds may also drain the pools of their amino acid precursors and thus increase the need for these amino acids in the diet. When amino acids are degraded for energy rather than converted to other compounds, the ultimate products become CO2, water, and urea. The CO2 and water are produced through classical pathways of intermediary metabolism involving the tricarboxylic acid (TCA) cycle. The urea is produced because other forms of waste N, such as ammonia (NH3), are toxic if their levels rise in the blood and inside cells. For mammals, urea production is a means of removal of waste N from the oxidation of amino acids in the form of a nontoxic, water-soluble compound.
More detailed descriptions of the amino acid pathways can be found in standard textbooks of biochemistry. Keep in mind when consulting such texts that pathways for nonmammalian systems (e.g., Escherichia coli and yeast) are often presented, and these pathways often have little importance to human biochemistry. When consulting reference material, the reader needs to be aware of the system of life from which the metabolic pathways and enzymes are being discussed. The discussion here is relevant to human biochemistry. Presented first is a discussion of the routes of degradation of each amino acid when the pathway is directed toward oxidation of the amino acid for energy. Next follows a discussion of pathways of amino acid synthesis, and finally the use of amino acids for other important compounds in the body is described.
Amino Acid Degradation Pathways
Complete amino acid degradation ends up with the production of N, which is removed by incorporation into urea. Carbon skeletons are eventually oxidized as CO2 via the TCA cycle (also known as the Krebs cycle or the citric acid cycle). The inputs to the cycle are acetyl-coenzyme A (CoA) and oxaloacetate forming citrate, which is degraded to α-ketoglutarate and then to oxaloacetate. The carbon skeletons from amino acid may enter the Krebs cycle via acetate as acetyl-CoA or via oxaloacetate/α-ketoglutarate. These latter two precursors are direct metabolites of the amino acids aspartate and glutamate. An alternative to the complete oxidation of the carbon skeletons to CO2 is the use of these carbon skeletons for the formation of fat and carbohydrate. Fat is formed from elongation of acetyl units, and so amino acids whose carbon skeletons degrade to acetyl-CoA and ketones may alternatively be used for synthesis of fatty acids. Glucose is split in glycolysis to pyruvate, and pyruvate is the immediate product of
alanine. Pyruvate may be converted back to glucose by elongation to oxaloacetate. Amino acids whose degradation pathways go toward formation of pyruvate, oxaloacetate, or α-ketoglutarate may be used for synthesis of glucose. Therefore, the degradation pathways of many amino acids can be partitioned into two groups with respect to the disposal of their carbon: amino acids whose carbon skeleton may be used for synthesis of glucose (gluconeogenic amino acids) or those whose carbon skeletons degrade for potential use for fatty acid synthesis.
alanine. Pyruvate may be converted back to glucose by elongation to oxaloacetate. Amino acids whose degradation pathways go toward formation of pyruvate, oxaloacetate, or α-ketoglutarate may be used for synthesis of glucose. Therefore, the degradation pathways of many amino acids can be partitioned into two groups with respect to the disposal of their carbon: amino acids whose carbon skeleton may be used for synthesis of glucose (gluconeogenic amino acids) or those whose carbon skeletons degrade for potential use for fatty acid synthesis.
The amino acids that degrade directly to the primary gluconeogenic and TCA cycle precursors, pyruvate, oxaloacetate, and α-ketoglutarate, do so by rapid and reversible transamination reactions:
L-glutamate + oxaloacetate ↔ α-ketoglutarate + L-aspartate
by the enzyme aspartate aminotransferase, which, of course, also can be
L-aspartate + α-ketoglutarate ↔ oxaloacetate + L-glutamate
and
L-alanine + α-ketoglutarate ↔ pyruvate + L-glutamate
by the enzyme alanine aminotransferase. What is quickly apparent is that the amino-N of these three amino acids may be rapidly exchanged and each amino acid rapidly converted to and from a primary compound of gluconeogenesis and the TCA cycle. As described later, compartmentation among different organ pools is the only limiting factor for complete and rapid exchange of the N of these amino acids.
The IDAAs leucine, isoleucine, and valine are grouped together under the heading of the BCAAs because the first two steps in their degradation pathway are common to all three amino acids:
The reversible transamination to keto acids is followed by irreversible decarboxylation of the carboxyl group to liberate CO2. The BCAAs are the only IDAAs that undergo transamination and therefore are unique among IDAAs.
Together, the BCAAs, alanine, aspartate, and glutamate make up the pool of amino-N that can move among amino acids via reversible transamination. As shown in Figure 1.2, glutamic acid is central to the transamination process. In addition, N can leave the transaminating pool by removal of the glutamate N via glutamate dehydrogenase, or it can enter by the reverse process. The amino acid glutamine is intimately tied to glutamate as well: all glutamine is made from amidation of glutamate, and glutamine is degraded by removal of the amide-N to form NH3 and glutamate.
 Fig. 1.2. Movement of amino-nitrogen (N) around glutamic acid. Glutamate undergoes reversible transamination with several amino acids. Nitrogen is also removed from glutamate by glutamate dehydrogenase, thus producing an α-ketoglutarate and an ammonia. In contrast, the enzyme glutamine synthetase adds an ammonia to glutamate to produce glutamine. Glutamine is degraded back to glutamate by liberation of the amide-N to release ammonia by a different enzymatic pathway (glutaminase). NH3, ammonia. |
A similar process occurs for formation and degradation of asparagine from aspartate. In terms of N metabolism, Figure 1.2 shows that the center of N flow in the body is through glutamate. This role becomes even clearer when we look at how urea is synthesized in the liver. The inputs into the urea cycle are a CO2, adenosine triphosphate (ATP), and NH3 to form carbamoyl phosphate, which condenses with ornithine to form citrulline (Fig. 1.3). The second N enters via aspartate to form argininosuccinate, which is then cleaved into arginine and fumarate. The arginine is hydrolyzed by arginase to ornithine, thus liberating urea. The resulting ornithine can reenter the urea cycle. As mentioned briefly later, some amino acids may liberate NH3 directly (e.g., glutamine, asparagine, and glycine), but most transfer through glutamate first, which is then degraded to α-ketoglutarate and NH3. The pool of aspartate is small in the body, and aspartate cannot be the primary transporter of the second N into urea synthesis. Rather, aspartate must act like arginine and ornithine as a vehicle for the introduction of the second N. If so, the second N is delivered by transamination via glutamate, again placing glutamate at another integral point in the degradative disposal of amino acid N.
An outline of the degradative pathways of the various amino acids is presented in Table 1.6. Rather than show individual reaction steps, the major pathways for degradation, including the primary end products, are presented. The individual steps may be found in current textbooks of biochemistry or in older reviews on the subject (13). Because of the importance of transamination, the majority of the N from amino acid degradation appears via N transfer to α-ketoglutarate to form glutamate. In some cases, the aminotransferase catalyzes the transamination reaction with glutamate bidirectionally, as indicated in Figure 1.2, and these enzymes are distributed in multiple tissues. In other cases, the transamination reactions are liver specific, are compartmentalized, and act specifically to degrade N, not reversibly exchange it. For example, when leucine labeled with the stable isotope tracer 15N was infused into dogs for 9 hours, considerable amounts of the 15N tracer were found in circulating glutamine + glutamate, alanine, the other two BCAAs, but not in tyrosine (14)—a finding indicating that the transamination of tyrosine did not proceed backward.
 Fig. 1.3. Urea cycle disposal of amino acid nitrogen (N). Urea synthesis incorporates one N from ammonia (NH3) and another from aspartate. Ornithine, citrulline, and arginine sit in the middle of the cycle. Glutamate is the primary source for the aspartate N; glutamate is also an important source of the ammonia into the cycle. ATP, adenosine triphosphate; CO2, carbon dioxide; NH2, amine. |
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Another reason that the entries in Table 1.6 do not show individual steps is that the specific pathways of the metabolism of all the amino acids are not clearly defined. For example, two pathways for cysteine are shown. Both are active, but how much cysteine is metabolized by which pathway is not as clear. Methionine is metabolized by conversion to homocysteine. The homocysteine is not directly converted to cysteine; rather, homocysteine condenses with a serine to form cystathionine, which is then broken apart to liberate cysteine, NH3, and ketobutyrate. The original methionine molecule appears as NH3 and ketobutyrate, however. The cysteine carbon skeleton comes from the serine. So the entry in Table 1.6 shows methionine degraded to NH3, yet this degradation pathway is the major synthesis pathway for cysteine. Because of the importance of the sulfur-containing amino acids, a more extensive discussion of the metabolic pathways of these amino acids may be found in a later chapter.
Glycine is degraded by more than one possible pathway, depending on the text used for reference. The primary pathway, however, appears to be the glycine cleavage enzyme system that breaks glycine into CO2 and NH3 and transfers a methylene group to tetrahydrofolate (15). This pathway has been shown to be the prominent pathway in rat liver and in other vertebrate species (16). Although this reaction degrades glycine, its importance is the production of a methylene group that can be used in other metabolic reactions.
Synthesis of Dispensable Amino Acids
The IDAAs are those amino acids that cannot be synthesized in sufficient amounts in the body and therefore must be in the diet in sufficient amounts to meet the body’s needs. Therefore, discussion of amino acid synthesis applies only to the dispensable amino acids. Dispensable amino acid synthesis falls into two groups: (a) amino acids that are synthesized by transferring an N to a carbon skeleton precursor that has come from the TCA cycle or from glycolysis of glucose and (b) amino acids that are synthesized specifically from other amino acids. Because this latter group of amino acids depends on the availability of other, specific amino acids, these amino acids are particularly vulnerable to becoming indispensable if the dietary supply of a precursor amino acid becomes limiting. In contrast, the former group is rarely rate limited in synthesis because of the ample precursor availability of carbon skeletons from the TCA cycle and from the labile amino-N pool of transaminating amino acids.
The pathways of dispensable amino acid synthesis are shown in Figure 1.4. As with amino acid degradation, glutamate is central to the synthesis of several amino acids by providing the N for synthesis. Glutamate, alanine, and aspartate may share amino-N transaminating back and forth among them (see Fig. 1.2). As Figure 1.4 is drawn, glutamate derives its N from NH3 with α-ketoglutarate, and that glutamate goes on to promote the synthesis of other amino acids. Kitagiri and Nakamura (17) argued that we have little capacity to form glutamate from NH3 and that the primary source of glutamate N comes from other amino acids via transamination. These amino acids ultimately result from dietary protein intake. Under circumstances of adequate dietary intake, the transaminating amino acids shown in Figure 1.2 supply more than adequate amino-N to glutamate. The transaminating amino acids act to provide a buffer pool of N that can absorb an increase in N from increased degradation or supply N when there is a drain. From this pool, glutamate provides material to maintain synthesis of ornithine and proline, of which proline is particularly important in protein synthesis of collagen and related proteins.
 Fig. 1.4. Pathways of the synthesis of dispensable amino acids. Glutamate is produced from ammonia (NH3) and α-ketoglutarate. That glutamate becomes the nitrogen source added to carbon precursors (pyruvate, oxaloacetate, glycolysis products of glucose, and glycerol) to form most of the other dispensable amino acids. Cysteine and tyrosine are different in that they require indispensable amino acid input for their production. |
Serine is produced from 3-phosphoglycerate that comes from glycolysis of glucose. Serine may then be used to produce glycine through a process that transfers a methylene group to tetrahydrofolate. This pathway is listed in Table 1.6 as a degradative pathway for serine, but it is also a source of glycine and one-carbon unit generation (15, 16). Conversely, this pathway actively operates backward to form serine from glycine in humans. When [15N]glycine is given orally, the primary transfer of 15N is to serine (18). Therefore, significant reverse synthesis of serine from glycine occurs. The other major place where 15N appears was in glutamate and glutamine, a finding indicating that the NH3 released by glycine oxidation is immediately picked up and incorporated into glutamate and the transaminating N-pool via glutamate dehydrogenase.
All the amino acids shown in Figure 1.4 have active routes of synthesis in the body (13), in contrast to the IDAAs for which no routes of synthesis exist in humans. This statement should be a simple definition of “indispensable” versus “dispensable.” In nutrition, however, we define a dispensable amino acid as an amino acid that is dispensable from the diet (3). This definition is different from defining the presence or absence of enzymatic pathways for an amino acid’s synthesis. For example, two of the dispensable amino acids depend on the degradation of IDAAs for their production: cysteine and tyrosine. Although serine provides the carbon skeleton and amino group of cysteine, methionine provides the sulfur through condensation of homocysteine and serine to form cystathionine (19). From the foregoing discussion, neither the carbon skeleton nor the amino group of serine is likely to be in short supply, but provision of sulfur from methionine may become limiting. Therefore, cysteine synthesis depends heavily on the availability of the IDAA methionine. The same is also true for tyrosine. Tyrosine is produced by the hydroxylation of phenylalanine, which is also the degradative pathway of phenylalanine. The availability of tyrosine strictly depends on the availability of phenylalanine and the liver’s ability to perform the hydroxylation.
Incorporation of Amino Acids into Other Compounds
Table 1.7 lists some of the compounds that amino acids are converted directly into or are used as important parts of the synthesis of other compounds in the body. The list is not inclusive, and it is meant to highlight important compounds in the body that depend on amino acids for their synthesis. Other important uses of amino acids are for the synthesis of taurine (20, 21) that is the “amino acid-like” 2-aminoethanesulfonate, found in far higher concentrations inside skeletal muscle than any amino acid (7). Another important, sulfur-containing compound is glutathione (22, 23, 24), a tripeptide composed of glycine, cysteine, and glutamate.
Carnitine (25) is important in the transport of longchain fatty acids across the mitochondrial membrane before fatty acids can be oxidized and is synthesized from ε-N,N,N-trimethyllysine (TML) (26). TML synthesis occurs from posttranslational methylation of specific lysine residues in specific proteins. TML is liberated when the proteins containing it are broken down (26). TML can also arise from hydrolysis of ingested meats. In contrast to 3-methylhistidine, TML can be found in proteins of both muscle and other organs such as liver (27). In rat muscle, TML is approximately one eighth as abundant as 3-methylhistidine.
TABLE 1.7 IMPORTANT PRODUCTS SYNTHESIZED FROM AMINO ACIDS | ||||||||||||||||||||||||||||||||||||||||||||||||
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Amino acids are the precursors for a variety of neurotransmitters that contain N. Glutamate may be an exception in that it serves both as a precursor for neurotransmitter production and is itself a primary neurotransmitter (28). Glutamate appears important in numerous neurodegenerative diseases from amyotrophic lateral sclerosis to Alzheimer disease. (29). Tyrosine is the precursor for catecholamine synthesis. Tryptophan is the precursor for serotonin synthesis. Various studies have reported the importance of plasma concentrations of these and other amino acids on the synthesis of their neurotransmitter products. The most common putative relationship cited is the administration of tryptophan, thus increasing brain serotonin levels.
Creatine and Creatinine
Most of the creatine in the body is found in muscle, where it exists primarily as creatine phosphate (30). When muscular work is performed, creatine phosphate provides the energy through hydrolysis of its “high-energy” phosphate bond that forms creatine with transfer of the phosphate to create an ATP. The reaction is reversible and is mediated
by the enzyme ATP-creatine transphosphorylase (also known as creatine phosphokinase).
by the enzyme ATP-creatine transphosphorylase (also known as creatine phosphokinase).
The original pathway of creatine synthesis from amino acid precursors was defined by Bloch and Schoenheimer in an elegant series of experiments using 15N-labeled compounds (31). Creatine is synthesized outside muscle in a two-step process (Fig. 1.5). The first step occurs in the kidney and involves the transfer of guanidino group of arginine onto the amino group of glycine to form ornithine and guanidinoacetate. Methylation of the guanidinoacetate occurs in the liver via S-adenosylmethionine to create creatine. Although glycine donates a N and carbon backbone to creatine, arginine must be available to provide the guanidino group, as well as methionine for donation of the methyl group. Creatine is then transferred to muscle, where creatine is phosphorylated. When creatine phosphate is hydrolyzed in muscle to form creatine, most of the creatine is recycled back to the phosphate form. A nonenzymatic process forming creatinine continually dehydrates some of the muscle creatine pool, however. Creatinine is not retained by muscle, but it is released into body water, is then removed by the kidney from blood, and is excreted into urine (32).
 Fig. 1.5. Synthesis of creatine and creatinine. Creatine is synthesized in the liver from guanidinoacetic acid, and that is synthesized in the kidney. Creatine taken up by muscle is primarily converted to phosphocreatine. Although there is some limited direct dehydration of creatine directly to creatinine, the majority of the creatinine comes from dehydration of phosphocreatine. Creatinine is rapidly filtered by the kidney into urine. ADP, adenosine diphosphate; ATP, adenosine triphosphate. |
The daily rate of creatinine formation is remarkably constant (≈1.7% of the total creatine pool per day) and depends on the mass of the creatine/creatine-phosphate pool, which is proportional to muscle mass (33). Thus, daily urinary output of creatinine has been used as a measure of total muscle mass in the body. Urinary creatinine excretion increases within a couple days after a creatine load has been added to the diet, and several more days are required after removal of creatine from the diet before urinary creatinine excretion returns to baseline—a finding indicating that creatine in the diet itself affects creatinine production (34). Therefore, consumption of creatine and creatinine in meat-containing foods increases urinary creatinine measurements. Although urinary creatinine measurements have been used primarily to estimate the adequacy of 24-hour urine collections, with adequate control of food composition and intake, creatinine excretion measurements are useful indices of body muscle mass (35, 36).
Purine and Pyrimidine Biosynthesis
The purines (adenine and guanine) and the pyrimidines (uracil, cytosine, and thymine) form the building blocks of DNA and RNA. Purines are heterocyclic double-ring compounds that require incorporation of two glutamine molecules (donation of the amide-N), a glycine molecule, a methylene group from tetrahydrofolate, and the amino-N of aspartic acid for their synthesis as inosine monophosphate. Adenine and guanine are formed from inosine monophosphate by the addition of another glutamine amide-N or aspartate amino-N.
Pyrimidines are synthesized after an amide-N of glutamine is condensed with a CO2 to form carbamoyl phosphate, which is further condensed with aspartic acid to make orotic acid, the pyrimidine’s heterocyclic 6-member ring. The enzyme that forms this carbamoyl phosphate is present in many tissues for pyrimidine synthesis, but it is not the enzyme found in the liver that makes urea (see Fig. 1.3). A block in the urea cycle causing a lack of adequate amounts of arginine to prime urea synthesis cycle in the liver, however, will result in diversion of unused carbamoyl phosphate to orotic acid and pyrimidine synthesis (37). Uracil is synthesized from orotic acid, and cytosine is synthesized from uracil by adding an amide group of glutamine to uridine triphosphate to form cytidine triphosphate.
TURNOVER OF PROTEINS IN THE BODY
As indicated earlier, proteins in the body are not static. Just as every protein is synthesized, it is also degraded. The concept that proteins are continually made and degraded in the body at different rates was first described by Schoenheimer and Rittenberg, who first applied isotopically labeled tracers of amino acids to the study of
amino acid metabolism and protein turnover in the 1930s. We now know that the rate of turnover of proteins in the body spans a broad range and that the rate of turnover of individual proteins tends to follow their function in the body; that is, those proteins whose concentrations need to be regulated (e.g., enzymes) or that act as signals (e.g., peptide hormones) have relatively high rates of synthesis and degradation as a means of regulating concentrations. Conversely, structural proteins such as collagen and myofibrillar proteins or secreted plasma proteins have relatively long lifetimes. Overall, however, a balance must exist between synthesis and breakdown of proteins. Balance in healthy adults who are neither gaining nor losing weight will be that the amount of N consumed as protein in the diet will match the amount of N lost in urine, feces, and other routes. Considerably, more protein is mobilized in the body every day than is consumed, however (Fig. 1.6).
amino acid metabolism and protein turnover in the 1930s. We now know that the rate of turnover of proteins in the body spans a broad range and that the rate of turnover of individual proteins tends to follow their function in the body; that is, those proteins whose concentrations need to be regulated (e.g., enzymes) or that act as signals (e.g., peptide hormones) have relatively high rates of synthesis and degradation as a means of regulating concentrations. Conversely, structural proteins such as collagen and myofibrillar proteins or secreted plasma proteins have relatively long lifetimes. Overall, however, a balance must exist between synthesis and breakdown of proteins. Balance in healthy adults who are neither gaining nor losing weight will be that the amount of N consumed as protein in the diet will match the amount of N lost in urine, feces, and other routes. Considerably, more protein is mobilized in the body every day than is consumed, however (Fig. 1.6).
Although no definable entity such as “whole body protein” exists, the term is useful for understanding the amount of energy and resources spent by the body in producing and breaking down protein in the body. Several methods using isotopically labeled tracers have been defined to quantitate the whole body turnover of proteins. An important point of Figure 1.6 is that the overall turnover of protein in the body is severalfold greater than the input of new dietary amino acids (38). A physiologically normal adult may consume 90 g of protein that is hydrolyzed and absorbed as free amino acids. Those amino acids mix with amino acids entering from protein breakdown from a variety of proteins. Approximately one third of the amino acids will appear from the large, but slowly turning over, pool of muscle protein. In contrast, considerably more amino acids will appear and disappear from proteins in the visceral and internal organs. These proteins make up a much smaller proportion of the total mass of protein in the body, but they have rapid synthesis and degradation rates. The overall result is that approximately 340 g of amino acids will enter the free pool daily, of which only 90 g will come from dietary amino acids. How do we assess in humans the turnover of protein in the body, however? These methods range from simple and noninvasive to expensive and complicated.
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