Biochemistry Book (Chapter 2 Amino Acids)

CHAPTER 2

Amino Acids

Proteins are the most abundant class of organic compounds in the healthy, lean human body, constituting more than half of its cellular dry weight. Proteins are polymers of amino acids and have molecular weights ranging from ap- proximately 10,000 to more than one million. Biochemical functions of proteins include catalysis, transport, contraction, protection, structure, and metabolic regulation.

Amino acids are the monomeric units, or building blocks, of proteins joined by a specific type of covalent linkage. The properties of proteins depend on the charac- teristic sequence of component amino acids, each of which has distinctive side chains.

Amino acid polymerization requires elimination of a water molecule as the carboxyl group of one amino acid reacts with the amino group of another amino acid to form a covalent amide bond. The repetition of this process with many amino acids yields a polymer, known as a polypeptide. The amide bonds linking amino acids to each other are known as peptide bonds. Each amino acid unit within the polypeptide is referred to as a residue. The sequence of amino acids in a protein is dictated by the sequence of nucleotides in a segment of the DNA in the chromosomes and the uniqueness of each living organism is due to its endowment of specific proteins.

2.1 L-(x-Amino Acids: Structure

Almost all of the naturally occurring amino acids in proteins are L-a-amino acids. The principal 20 amino acids in

proteins have an amino group, a carboxyl group, a hydrogen atom, and an R-group attached to the a-carbon (Figure 2-1). Proline is an exception because it has a cyclic structure and contains a secondary amine group (called an imino group) instead of a primary amine group (called an amino group). Amino acids are classified according to the chemical properties of the R-group. Except for glycine (R = H), the amino acids have at least one asymmetrical carbon atom (the a-carbon). The absolute configuration of the four groups attached to the a-carbon is conventionally compared to the configuration of L-glyceraldehyde (Figure 2-2). The D and L designations specify absolute configuration and not the dextro (right) or levo (left) direction of rotation of plane- polarized light by the asymmetrical carbon center. In organic chemistry, the assignment of absolute configurations of an asymmetrical center is made by the R and S classification of isomers. This system prioritizes the substituents linked to the asymmetrical carbon atom (e.g., decreasing atomic number or valence density) and the assignment is based upon the clockwise (R) or the counterclockwise (S) positioning of the three higher priority groups.

2.2 Classification

A useful classification of the amino acids is based on the solubility (i.e., ionization and polarity) characteristics of the side chains (R-groups). The R-groups fall into four classes:

17

18 CHAPTER 2 Amino Acids

COOH (~-Carbon

I R

FIGURE 2-1 Basic structure of an or-amino acid.

1. Nonpolar (hydrophobic), 2. Polar, negatively charged (acidic), 3. Polar, positively charged (basic), and 4. Polar, neutral (un-ionized).

Within each class, R-groups differ in size, shape, and other properties. Figure 2-3 shows the structure of each amino acid according to this classification with the R- group outlined. Ionizable structures are drawn as they would exist at pH 7.0. The three-letter and one-letter abbreviations for each amino acid are given in Table 2-1. A -yl ending on amino acid residue indicates that the carboxyl group of an amino acid is linked to another functional group (e.g., in a peptide bond).

The eight essential amino acids (Table 2-1) are those which humans cannot synthesize and which must be supplied in the diet. The remaining amino acids are synthesized in the body by various biochemical pathways (Chapter 17).

Nonpolar Amino Acids

Glycine

Glycine is the smallest amino acid and has an H atom as its R-group. It is the only u-amino acid that is not optically active. The small R-group provides a mini- mum of steric hindrance to rotation about bonds; therefore, glycine fits into crowded regions of many peptide chains. Collagen, a rotationally restricted fibrous protein, has glycyl residues in about every third position in its polypeptide chains. Glycine is used for the biosynthesis of many nonprotein compounds, such as porphyrins and purines.

Glycine and taurine are conjugated with bile acids, products derived from cholesterol, before they are excreted into the biliary system. Conjugated bile acids are amphipathic and are important in lipid absorption (Chapter 12). Glycine also is a neurotransmitter; it is inhibitory in the spinal cord and excitatory in the cerebral cortex and other regions of the forebrain. Nonketotic hyperglycinemia (NKH) is an in- born error of glycine degradation in which a large amount of glycine accumulates throughout the body. NKH causes

CHO CHO

OH H H OH

CH2OH CH2OH

CHO CHO I I

H~C~OHI O H - - ? ~ H

CH2OH CH2OH

D-Glyceraldehyde L-Glyceraldehyde

CHO CHO �9

�9

H"- C--- OH H 0 - - C.,--'.H �9 �9

CH,OH CH,OH Fischer perspective formulas

CHO CHO

OH HO ....... I H

CH2OH CH2OH

Fischer projection formulas

COOH COOH �9 ~

�9

H--C-=" NH2 H,N-- r ~

(-,H3 CH3 D-Alanine L-Alanine

FIGURE 2-2 Different representations of the configurational stereoisomers of glyceraldehyde and of alanine.

severe consequences in the central nervous system (CNS) and leads to death (Chapter 17).

CO0 - I

+H3N--C--H I H

Glycine

SECTION 2.2 Classification 19

FIGURE 2-3 Classification of commonly occurring L-a-amino acids based on the polarity of side chains (R-groups) at pH 7.0.


TABLE 2-1 Amino Acid Abbreviations and Nutritional Property

Amino Abbreviation Designation Nutritional acid letter property*

Alanine Ala Arginine Arg

Asparagine Asn Aspartic acid Asp Cysteine Cys Glutamic acid Glu Glutamine Gln

Glycine Gly Histidine His

Isoleucine lie Leucine Leu Lysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine Ser Threonine Thr Tryptophan Trp Tyrosine Tyr Valine Val

A non-essential R conditionally

essential N non-essential D non-essential C non-essential E non-essental Q conditionally

essential G non-essential H conditionally

essential I essential L essential K essential M essential F essential P non-essential S non-essential T essential W essential Y non-essential V essential

*The eight essential amino acids are not synthesized in the body and have to be supplied in the diet. The conditionally essential amino acids, although synthesized in the body, may require supplementation during certain physiological conditions such as pregnancy. The non-essential amino acids can be synthesized from various metabolites.

Alanine

The side chain of alanine is a hydrophobic methyl group,--CH3. Other amino acids may be consid- ered to be chemical derivatives of alanine, with substituents on the fl-carbon. Alanine and glutamate provide links between amino acid and carbohydrate metabolism (Chapter 22).

COO - I

+H3N~C~H

,~CarrbOn.~Ci H 3

A l a n i n e

Valine, Leucine, and Isoleucine

These branched-chain aliphatic amino acids contain bulky nonpolar R-groups and participate in hydrophobic interactions. All three are essential amino acids. A de- fect in their catabolism leads to maple syrup urine disease (Chapter 17). Isoleucine has asymmetrical centers at both the o~- and/3-carbons and four stereoisomers, only one of which occurs in protein. The bulky side chains tend to as- sociate in the interior of water-soluble globular proteins. Thus, the hydrophobic amino acid residues stabilize the three-dimensional structure of the polymer.

COO- COO- l I

COO- +H3N~C~H +H3N~C~H I I I

+H3N~C~H CH 2 H~C~CH 3 I I I CH CH CH 2

/ \ / \ i H3C OH 3 H3C OH3 OH 3

Valine Leucine Isoleucine

Phenylalanine

A planar hydrophobic phenyl ring is part of the bulky R-group of phenylalanine. It is an essential amino acid whose metabolic conversion to tyrosine is defective in phenylketonuria (Chapter 17). Phenylalanine, tyrosine, and tryptophan are the only a-amino acids that contain aromatic groups and consequently are the only residues that absorb ultraviolet (UV) light (Figure 2-4). Trypto- phan and tyrosine absorb significantly more energy than phenylalanine at 280 nm, the wavelength generally used to measure the concentration of protein in a solution.

CO0 - I

+H3N~C~H I

P h e n y l a l a n i n e

Tryptophan

A bicyclic nitrogenous aromatic ring system (known as an indole ring) is attached to the /~-carbon of alanine to form the R-group of tryptophan. Tryptophan is a precursor of serotonin, melatonin, nicotinamide, and many


FIGURE 2-4 UV absorption spectra of phenylalanine, tyrosine, and tryptophan.

naturally occurring medicinal compounds derived from plants. It is an essential amino acid. The indole group absorbs UV light at 280 n m - - a property that is useful for spectrophotometric measurement of protein concentration (Figure 2-4).

Tryptophan and tyrosine both show fluorescence; however, tryptophan absorbs more intensely. When molecules are raised to a higher energy state by absorption of radiant energy, they generally are unstable and return to the ground state. The energy released in this process manifests as heat (radiation energy) or light. The process of light emission is known as fluorescence. The quantum of energy re-emitted as fluorescence is always less than that of absorbed energy. Thus, the fluorescent light always appears at a longer wavelength (lower energy) than the original absorbed light energy (Figure 2-5). Tryptophan fluorescence studies can provide valuable in- formation regarding protein and protein-ligand confor- mations due to the effects of surrounding amino acid residues.

COO -

I + H3N-- -C- - - H

I CH2 I

N/CH

Tryp tophan

Methionine

This essential amino acid contains an R-group with a methyl group attached to sulfur. Methionine serves as donor of a methyl group in many transmethylation reactions, e.g., in the synthesis of epinephrine, creatine, and

FIGURE 2-5 Tryptophan fluorescence spectrum. The emission spectrum appears at longer wavelengths as compared to the absorption spectrum.

melatonin. Almost all of the sulfur-containing compounds of the body are derived from methionine.

COO -

I +H3N~C---H

I CH2 I

CH2 I S I CH3

Methionine

Proline

Proline contains a secondary amine group, called an imine, instead of a primary amine group. For this reason, proline is called an imino acid. Since the three-carbon R-group of proline is fused to the ol-nitrogen group, this compound has a rotationally constrained rigid-ring structure. As a result, prolyl residues in a polypeptide introduce restrictions on the folding of chains. In collagen, the principal protein of human connective tissue, certain prolyl residues are hydroxylated (Figure 2-6). The hydroxylation occurs during protein synthesis and requires ascorbic acid (vitamin C) as a cofactor. Deficiency of vitamin C causes formation of defective collagen and scurvy (Chapters 25 and 38).

COO -

I +H2N C~H

! I H2C /OH2

~ C H 2

Proline


1COOH

HN" 2~C--H

H2C~ 4 ~CH2 ~ C ~

H OH 4-Hydroxyproline

Present in collagen, a fibrous protein.

COOH I

H2N- -C- -H I

CH2 I

CH~_ I

CH2 I

ECH 2 I N

H ~CH3 ~-N-Methyllysine

The N-trimethyl derivative of lysine is involved in the synthesis of carnitine.

COOH

H2N--C- -H

CH2

CH2

HC--OH

CH2

NH2 5-Hydroxylysine

COO - I

H N- - - - - - -C- - H I I

O---G~ ~CH2 C H2

Pyrrolidone car boxylate (pyroglutamate)

Present in some proteins and peptides as N-terminal amino acid residue.

COOH I

H2N--C- -H I

CH2 I

C -C I I

H 3 C - - N ~ ~ N

H a-Methylhistidine

Present in myosin, a muscle protein.

COOH I

H2N--C- -H I

CH2 I

CH ,OOC "~ ~COOH

7-Carboxyglutamic acid

COOH I

H 2 N - - C - - H I CH2 I O I

HO--P--OH II O

O-Phosphoserine

Phosphorylation and dephosphorylation of selected serine residues in a variety of proteins play an important role in the regulation of metabolism and are mediated by some hormones.

COOH I

H=N--C--H I CH=

r t

CH2 , I

(H3C),N--C--H I

H=N ~ C ~ o Diphthamide

A novel derivative of histidine present only in the eukaryotic protein elongation factor 2 (EF-2), which participates in the elongation step of

protein biosynthesis. Diphtheria toxin inhibits eukaryotic protein synthesis by catalyzing a covalent modification of diphthamide (see Chapter 25).

Present in certain blood-clotting proteins

H2N ~ /COOH

c~

H2N~ 4z=.~ ~

HOoc/CH-- (CH,)~ I

;H~ 3

f N H 2

~ 3 (CRy#__ CH ~ C O O H

,H~,

H 2 N f C % o o H Desmosine

NH2

HOOc./CH -- (CH=) (OH=)E-- CH --COOH

(CH=)F-- CH--COOH 'I" I NH2 (?H=),

H ~ C % o o H Isodesmosine

Desmosine and isodesmosine are formed from lysyl residues of the polypeptide chains of elastin, a fibrous protein.

FIGURE 2-6 Modified derivatives of certain amino acids are found in proteins.


Acidic Amino Acids

Aspartic Acid

The ]3-carboxylic acid group of aspartic acid has a pK' of 3.86 and is ionized at pH 7.0 (the anionic form is called aspartate). The anionic carboxylate groups tend to occur on the surface of water-soluble proteins, where they inter- act with water. Such surface interactions stabilize protein folding.

COO - I

+ H3N---~C~H I

~CH2 I

Aspartate

Glutamic Acid

The v-carboxylic acid group of glutamic acid has a pK' of 4.25 and is ionized at physiological pH. The anionic groups of glutamate (like those of aspartate) tend to occur on the surfaces of proteins in aqueous environments. Glutamate is the primary excitatory neurotransmitter in the brain. Its levels are regulated by clearance that is mediated by glutamate transfer protein in critical motor control areas in the CNS. In amyotrophic lateral sclerosis (ALS) glutamate levels are elevated in serum, spinal fluid, and brain; glutamate excitotoxicity is implicated in the progression of the disease. ALS is a progressive disorder affecting motor neurons in the spinal cord, brain stem, and cortex. The pre- cise molecular basis of the disease is unknown; however, factors involved are glutamate excitotoxicity, genetics, ox- idative stress, and diminished neurotrophic factors.

Two drugs that provide neuroprotection against glutamate excitotoxicity are riluzole and gabapentin. Gabapentin is an amino acid structurally related to the neurotransmitter v-aminobutyrate (GABA). GABA, an inhibitory neurotransmitter in the CNS, is produced by the decarboxylation of glutamate by glutamate de- carboxylase, a pyridoxal phosphate dependent enzyme. GABA, when bound to its receptors, causes an increase in permeability to chloride ions in neuronal cells. A group of tranquilizing drugs known as benzodiazopines enhance the membrane permeability of chloride ions by GABA. In some proteins, the v-carbon of glutamic acid contains an additional carboxyl group. Residues of v-carboxyglutamic acid (Gla) bear two negative charges and can strongly bind calcium ions. v-Carboxylation of glutamic acid residues is a posttranslational modification and requires vitamin K as a cofactor, v-Carboxyglutamate

residues are present in a number of blood coagulation proteins (factors II, VII, IX, and X) and anticoagulant proteins C and S (Chapter 36). Osteocalcin, a protein present in the bone, also contains v-carboxyglutamate residues (Chapter 37).

A cyclic, internal amide derivative of glutamic acid is pyrrolidone carboxylic acid (also known as pyroglu- tamic acid or 2-oxoproline). Some proteins (e.g., heavy chains of immunoglobulins; Chapter 35) and peptides (e.g., thyrotropin-releasing hormone; Chapter 33) have py- roglutamic acid as their N-terminal amino acid residue.

COO -

I COO - +H3N--(zC--H

I I ~CH2 HN C - - H

I I I yCH2 O---C OH2

I ~ c ~ H2

O / / ' C ~ o - Pyrrolidone carboxylate Glutamate (pyroglutamate)

COO - I NH2

+H3NmCmH I I CH2 CH 2 I I CH2

CH I I CH2

I O - - c / C ~ c = o COO-

l I },-amino butyrate O- O- (GABA)

y -Carboxy glutamate

Basic Amino Acids

Lysine

Lysine is an essential amino acid. The long side chain of lysine has a reactive amino group attached to the s-carbon. The s-NH2 (pK'= 10.53) is protonated at physiological pH. The lysyl side chain forms ionic bonds with negatively charged groups of acidic amino acids. The s-NH2 groups of lysyl residues are covalently linked to biotin (a vitamin), lipoic acid, and retinal, a derivative of vitamin A and a constituent of visual pigment.

In collagen and in some glycoproteins, g-carbons of some lysyl residues are hydroxylated (Figure 2-6), and sugar moieties are attached at these sites. In elastin and collagen, some s-carbons of lysyl residues are oxidized to reactive aldehyde (-CHO) groups, with elimination of NH3. These aldehyde groups then react with other s - NH2 groups to form covalent cross-links between polypeptides, thereby providing tensile strength and insolubility to


protein fibers. Examples of cross-linked amino acid structures are desmosine, isodesmosine (Figure 2-6), dehy- drolysinonorleucine, lysinonorleucine, merodesmosine, and dehydromerodesmosine (Chapter 10). Lysyl R-groups participate in a different type of cross-linking in the formation of fibrin, a process essential for the clotting of blood. In this reaction, the s-NH2 group of one fibrin polypeptide forms a covalent linkage with the glutamyl residue of another fibrin polypeptide (Chapter 36).

COO -

+H3N~=C~H

/YCH2

yCH2

6CH2

sCH2

NH3 + Lysine

Histidine

The imidazole group attached to the/~-carbon of histidine has a pK' value of 6.0. The pK' value of his- tidyl residues in protein varies depending on the nature of the neighboring residues. The imidazolium-imidazole buffering pair has a major role in acid-base regulation (e.g., hemoglobin). The imidazole group functions as a nucleophile, or a general base, in the active sites of many enzymes and may bind metal ions. Histidine is nonessen- tial in adults but is essential in the diet of infants and indi- viduals with uremia (a kidney disorder). Decarboxylation of histidine to yield histamine occurs in mast cells present in loose connective tissue and around blood vessels, ba- sophils of blood, and enterochromaffin-like (ECL) cells present in the acid-producing glandular portion (oxyntic cells) of the stomach.

The many specific reactions of histamine are deter- mined by the type of receptor (HI or H2) present in the target cells. The contraction of smooth muscle (e.g., gut and bronchi) is mediated by Hi receptors and antagonized

COO - H I , I

+H3N--C~H H 3 N ~ C ~ H I I CH2 CH2 I I C CH C CH I I I I N,. NH N,. NH ~ c / "~c j

H H Histidine Histamine

by diphenhydramine and pyrilamine. Hi-receptor antagonists are used in the treatment of allergic disorders. Secre- tion of HC1 by the stomach (Chapter 12) and an increase in heart rate are mediated by H2 receptors. Examples of Hz-receptor antagonists of histamine action are cimeti- dine and ranitidine, agents used in the treatment of gastric ulcers.

Arginine

The positively charged guanidinium group attached to the 6-carbon of arginine is stabilized by resonance between the two NH2 groups and has a pK' value of 12.48. Arginine is utilized in the synthesis of creatine and it participates in the urea cycle (Chapter 17).

The nitrogen of the guanidino group of arginine is converted to nitric oxide (NO) by nitric oxide synthase. NO is unstable, highly reactive, and has a life span of only a few seconds. However, NO affects many biological activities, including vasodilation, inflammation, and neurotransmis- sion (Chapter 17).

COO- l

+H3N--CmH I CH2 I CH2 I

6CH2 I NH I C~'NH2 ~ Guanidinium I , NH~. . . . . . ! group

Arginine

N e u t r a l A m i n o A c i d s

Serine

The primary alcohol group of serine can form esters __ . : * . L . _1_ I . . . . ." _ ~ _ ". _1 [ 11 ~ ' . . . . . . . , ~ l f ' ~ ~ . _ _1 _ 1_ _ "_ _1 . . . . . : a_ l _

w i t . p . u ~ p . u l l ~ a~lu t l " l gu l c z.-U) al lu g ly~u~luc~ wluJ

C O O -

I +H3NmC--H

I H m C - - O H

I H

Serine

sugars. The phosphorylation and dephosphorylation pro- cesses regulate the biochemical activity of many proteins. Active centers of some enzymes contain seryl hydroxyl


groups and can be inactivated by irreversible derivatiza- tion of these groups. The-OH group of serine has a weakly acidic pK' of 13.6.

Threonine This essential amino acid has a second asymmetrical

carbon atom in the side chain and therefore can have four isomers, only one of

CO0 - I

+H3N~C~H I

H ~ C ~ O H I CH3

Threonine

which, L-threonine, occurs in proteins. The hydroxyl group, as in the case of serine, participates in reactions with phosphoric acid and with sugar residues.

Cysteine The weakly acidic (pK'= 8.33) sulfhydryl group (-SH)

of cysteine is essentially undissociated at physiological pH. Free-SH groups are essential for the function of many enzymes and structural proteins. Heavy metal ions, e.g., Pb 2+ and Hg 2+, inactivate these proteins by combining with their-SH groups. Two cysteinyl-SH groups can be oxidized to form cystine. A covalent disulfide bond of cystine can join two parts of a single polypeptide chain or two different polypeptide chains through cross-linking of cysteine residues. These - S - S - bonds are essential both for the folding of polypeptide chains and for the association of polypeptides in proteins that have more than one chain, e.g., insulin and immunoglobulins.

CO0 - I

+H3N~C~H I CH2 I SH

Cyste ine

CO0 - I

+H3N~C~H I C H 2 ~ S ~ S

Cystine

COO- I

+H3N~C~H I CH2

Tyrosine The phenolic hydroxyl group of this aromatic amino

acid has a weakly acidic pK' of about 10 and therefore is un-ionized at physiological pH. In some enzymes, the hydrogen of the phenolic hydroxyl group can participate in hydrogen bond formation with oxygen and nitrogen atoms. The phenolic hydroxyl group of tyrosine residues in protein can be sulfated (e.g., in gastrin and chole- cystokinin; see Chapter 12) or phosphorylated by a reaction catalyzed by the tyrosine-specific protein kinase that is a product of some oncogenes (Chapter 26). Tyro- sine kinase activity also resides in a family of cell surface receptors that includes receptors for such anabolic polypeptides as insulin, epidermal growth factor, platelet- derived growth factor, and insulin-like growth factor type 1. All of these receptors have a common motif of an ex- ternal ligand binding domain, a transmembrane segment, and a cytoplasmic tyrosine kinase domain (Chapter 22). Tyrosine accumulates in tissues and blood in tyrosinosis and tyrosinemia, which are due to inherited defects in catabolism of this amino acid. Tyrosine is the biosynthetic precursor of thyroxine, catecholamines, and melanin. Tyrosine and its biosynthetic precursor, phenylalanine, both absorb UV light (Figure 2-4).

COO - I

+H3N--C--H I CH2

C) OH

Tyrosine

Asparagine The R-group of this amide derivative of aspartic acid has

no acidic or basic properties but is polar and participates in hydrogen bond formation. It is hydrolyzed to aspartic acid and ammonia by the enzyme asparaginase. In glycoproteins, the carbohydrate side chain is often linked through the amide group of asparagine.

COO -

I +H3N~C~H

I CH2 I

O// 'C~NH 2 Asparagine

2fi CHAPTER 2 Amino Acids

Glutamine

This amide of glutamic acid has properties similar to those of asparagine. The 7-amido nitrogen, derived from ammonia, can be used in the synthesis ofpurine and pyrim- idine nucleotides (Chapter 27), converted to urea in the liver (Chapter 17), or released as NH3 in the kidney tubu- lar epithelial cells. The last reaction, catalyzed by the enzyme glutaminase, functions in acid-base regulation by neutralizing H + ions in the urine (Chapter 39).

Glutamine is the most abundant amino acid in the body. It composes more than 60% of the free amino acid pool in skeletal muscle. It is metabolized in both liver and gut tissues. Glutamine, along with alanine, are significant precursors of glucose production during fasting (Chapter 15). It is a nitrogen donor in the synthesis of purines and pyrimidines required for nucleic acid synthesis (Chapter 27). Glutamine is enriched in enteral and parenteral nutrition to promote growth of tissues; it also enhances immune functions in patients recovering from surgical procedures. Thus, glutamine may be classified as a conditionally essential amino acid during severe trauma and illness.

CO0 - I

+H3N---C~H I

CH2

I CH2 I

O~/ 'C~NH 2 Glutamine

Unusual Amino Acids

Several L-amino acids have physiological functions as free amino acids rather than as constituents of proteins. Exam- ples are as follows:

1. ~-Alanine is part of the vitamin pantothenic acid. 2. Homocysteine, homoserine, omithine, and citrulline

are intermediates in the biosynthesis of certain other amino acids.

3. Taurine, which has an amino group in the ~-carbon and a sulfonic acid group instead of COOH, is present in the CNS and as a component of certain bile acids participates in digestion and absorption of lipids in the gastrointestinal tract.

4. y-Aminobutyric acid is an inhibitory neurotransmitter.

5. Hypoglycin A is present in unripe akee fruit and produces severe hypoglycemia when ingested.

6. Some D-amino acids are found in polypeptide antibiotics, such as gramicidins and bacitracins, and in bacterial cell wall peptides.

Amino Acids Used as Drugs

D-Penicillamine (D-fl,/3-dimethylglycine), a metabolite of penicillin, was first isolated in the urine specimens from patients treated with penicillin with liver disease. It is an ef- fective chelator of metals such as copper, zinc, and lead. It is used in the chelation therapy of Wilson's disease, which is characterized by excess copper accumulation leading to hepatolenticular degeneration (Chapter 37).

CH 3 I

H 3 C ~ C ~ CH - - CO0" I I SH NH +

D - p e n i c i l l a m i n e

N-Acetylcysteine is administered in the ace- toaminophen toxicity. It replenishes the hepatic stores of glutathione (Chapter 17). N-Acetylcysteine is also used in the treatment of pulmonary diseases including cystic fibrosis (Chapter 12). In patients with chronic renal insufficiency, prophylactic oral administration of N-Acetylcysteine have been used in the prevention of further renal impairment due to administration of radiographic contrast agents. In this setting presum- ably N-Acetylcysteine functions as an antioxidant and augments the vasodilatory effect of nitric oxide via the formation of S-nitrosothiol (Chapter 17).

COOH O I II

H S ~ C H 2 - - - C H - - N H ~ C ~ C H 3

N-acetylcysteine

Gabapentin is y-aminobutyrate covalently linked to cy- clohexane to make it lipophilic and to facilitate its transport across the blood-brain barrier. It is used as an anticonvul- sant and in amyotrophic lateral sclerosis (ALS).

H 3 N ' ~ C O O

Gabapentin

SECTION 2.3 Electrolyte and Acid-Base Properties 27

2.3 Electrolyte and Acid-Base Properties

Amino acids are ampholytes, i.e., they contain both acidic and basic groups. Free amino acids can never occur as neutral nonionic molecules:

H 0 I II

R--C--C--OH I NH2

Instead, they exist as neutral zwi t t e r ions that contain both positively and negatively charged groups:

H / o Nonpolar I neutral = R- -C- -CI R-group I ~ " 0

NH3

Ammonium cation Zwitterion

e Resonance-

,-- stabilized carboxylate anion

Zwitterions are electrically neutral and so do not migrate in an electric field. In acidic solution (below pH 2.0), the predominant species of an amino acid is positively charged and migrates toward the cathode:

H O I II

R--C--C--OH I NH3 +

FIGURE 2-7 Titration profile of glycine, a monoaminomonocarboxylic acid.


In basic solution (above pH 9.7), the predominant species is negatively charged and migrates toward the anode:

H O I H

R m C _ C _ O -

I NH2

The isoelectric point (pI) of an amino acid is the pH at which the molecule has an average net charge of zero and therefore does not migrate in an electric field. The pI is calculated by averaging the pK' values for the two functional groups that react as the zwitterion becomes alternately a monovalent cation or a monovalent anion.

At physiological pH, monoaminomonocarboxylic amino acids, e.g., glycine and alanine, exist as zwitterions.

FIGURE 2-8 Titration profile of histidine.

SECTION 2.3 Electrolyte and Acid-Base Properties 29

That is, at a pH of 6.9-7.4, the oe-carboxyl group (pK' 2.4) is dissociated to yield a negatively charged carboxylate ion (-COO-), while the ol-amino group (pK' 9.7) is protonated to yield an ammonium group (-NH~-). The pK' value of the o~-carboxyl group is considerably lower than that of a comparable aliphatic acid, e.g., acetic acid (pK' 4.6). This stronger acidity is due to electron withdrawal by the positively charged ammonium ion and the con- sequent increased tendency of a carboxyl hydrogen to dissociate as an H +. The or-ammonium group is corre-

spondingly a weaker acid than an aliphatic ammonium ion, e. g., ethylamine (pK' 9.0) because the inductive effect of the negatively charged carboxylate anion tends to prevent dissociation of H +. The titration profile of glycine hydrochloride (Figure 2-7) is nearly identical to the profiles of all other monoaminomonocarboxylic amino acids with nonionizable R-groups (Ala, Val, Leu, Ile, Phe, Ser, Thr, Gln, Asn, Met, and Pro).

The titration of glycine has the following major fea- tures. The titration is initiated with glycine hydrochloride,

FIGURE 2-9 Titration profile of lysine.


F I G U R E 2-11} Titration profile of aspartic acid.

CI-(H~-NCH2COOH), which is the fully protonated form of the amino acid. In this form, the molecule contains two acidic functional groups; therefore, two equivalents of base are required to completely titrate 1 mol of glycine hydrochloride. There are two pK' values: pK' 1 due to reaction of the carboxyl group and pK~ due to reaction of the ammonium group. Addition of 0.5 eq of base to 1 mol of glycine hydrochloride raises the pH 2.34 (PKtl), whereas addition of 1.5 eq further increases the pH to 9.66 (pK~). At low pH values (e.g., 0.4), the molecules are

predominantly cations with one positive charge; at pH values of 5-7, most molecules have a net charge of zero; at high pH values (e.g., 11.7), all of the molecules are essentially anions with one negative charge. The midpoint between the two pK' values [i.e., at pH = (2.34 + 9.66)/2 = 6.0] is the pI. Thus, pI is the arithmetic mean of pK' 1 and pK~ values and the inflection point between the two seg- ments of the titration profile.

The buffering capacities of weak acids and weak bases are maximal at their pK values. Thus, monoaminomono-

SECTION 2.4 Chemical Reactions of Amino Acids 31

TABLE 2-2 pK'and p l Values of Selected Free Amino Acids at 25~ *

Amino Acid pK' 1 (a-COOH) pK' 2 pK' 3 pI

Alanine 2.34 9.69 (a-NH3+)

Aspartic acid 2.09 3.86 (/3-COOH) 9.82 (a-NH 3 +)

Glutamic acid 2.19 4.25 (7-COOH) 9.67 (tx-NH3 +)

Arginine 2.17 9.04 (a-NH3+) 12.48 (Guanidinium)

Histidine 1.82 6.00 (Imidazolium) 9.17 (NH3+)

Lysine 2.18 8.95 (a-NH3+) 10.53 (e-NH3 +)

Cysteine 1.71 8.33 (-SH) 10.78 (~-NH3+)

Tyrosine 2.20 9.11 (a-NH3 +) 10.07 (Phenol OH)

Serine 2.21 9.15 (a-NH3 +) 13.6 (Alcohol OH)

6.00

298 (pK I+pK21) 2 3.22 ( pK'I +2pK'2)

pK' + PK'3) 10.76 2

(eKe+eKe) 7.59 2

(PK'2 + PK'3) 9.74 2

5.02 ( pK'I +2 pK'2 )

5.66 ( pK'I +2 pK'2 )

5.68 ( pK'I +2 pK'2)

*The pK' values for functional groups in proteins may vary significantly from the values for free amino acids.

carboxylic acids exhibit their greatest buffering capacities in the two pH ranges near their two pK' values, namely, pH 2.3 and pH 9.7 (Figure 2-7). Neither these amino acids nor the a-amino or ot-carboxyl groups of other amino acids (which have similar pK' values) have significant buffering capacity in the neutral (physiological) pH range. The only amino acids with R-groups that have buffering capacity in the physiological pH range are histidine (imidazole; pK' 6.0) and cysteine (sulfhydryl; pK' 8.3). The pK' values for R-groups vary with the ionic environment. The titration profile of histidine is shown in Figure 2-8. The pI is the mean of pK~ and pKg.

Titration profiles of the basic and acidic amino acids lysine and aspartic acid are shown in Figures 2-9 and 2-10. The R-groups are ionized at physiological pH and have anionic and cationic groups, respectively. The pI value for aspartic acid is the arithmetic mean of pK' 1 and pK~, whereas for lysine and histidine the pI values

are given by the arithmetic mean of pK~ and pKg. The pK' and pI values of selected amino acids are listed in Table 2-2.

2.4 Chemical Reactions of Amino Acids

The reactions of amino acids with ninhydrin, carbon dioxide, metal ions, and glucose are described below. The last three are of physiological importance.

Ninhydrin (triketohydrindene hydrate) reacts with o~- amino acids to produce CO2, NH3, and an aldehyde with one less carbon than the parent amino acid. In most cases, a blue or violet compound (proline and hydroxyproline give a yellow color) is formed owing to reaction of the liberated NH3 with ninhydrin, as shown in Figure 2-11. Color and CO2 production provide a basis for the quantitative determination of amino acids.


O II C\ /OH

7 ~ O H II o

Ninhydrin

H I

R m C R C O O H I

NH2

Amino acid

H

+ CO2 + H20

O II

7 ~NH 2 I! o o II

II o

Ninhydrin

o o - I! I QL c T = N - \ § 2.20 II II o o Purple-colored ion

FIGURE 2-11 Reaction of an o~-amino acid with ninhydrin. Two molecules of ninhydrin and the nitrogen atom of the amino acid are involved in the production of the purple product.

Ammonia, some amines, and some proteins and peptides will also yield a colored product but will not gen- erate CO2. Thus, the colorimetric analysis is not specific for amino acids unless CO2 release is measured or the amino acids are purified and freed from interfering materials (the usual procedure). The color reaction with ninhydrin is used extensively in manual and automated procedures.

CO2 adds reversibly to the un-ionized amino group of an amino acid. The product is a carbamate (or carboxyamino) derivative.

0 0

C/LO - H C l[110-

~ C H2NaC--H = N--C--H I I

j R O / / ,C .o _R

+ H +

This type of reaction accompanies transport of C02 in the blood (Chapter 1). In tissue capillaries, CO2 combines with free o~-amino groups of hemoglobin to form carbaminohemoglobin; in pulmonary capillaries, this reaction is reversed to release CO2 into the alveoli. This mode of transport is limited to only about 10% of the carbon dioxide transported in the blood.

Metal ions can form complexes with amino acids. Metal ions that function in enzymatic or structural biochemical systems include those of iron, calcium, copper, zinc, magnesium, cobalt, manganese, molybdenum, nickel, and chromium. The functional group that binds a metal ion is called a ligand. Ligands are electron donors that form non- covalent bonds with the metal ions, usually two, four, or six ligand groups per ion. When four ligand groups bind a metal ion, the complex is either a plane or a tetrahedron; when six ligand groups participate, octahedral geometry results. The term chelation is applied to a metal-ligand interaction when a single molecule provides two or more ligands (e.g., chelation of iron with four nitrogens in one porphyrin molecule; see Chapter 14).

Metal ions can also react with amino acid functional groups to abolish the biological activity of proteins. Heavy metal ions that form highly insoluble sulfides (e.g., HgS, PbS, CuS, AgzS) characteristically react with sulfhydryl groups of cysteinyl residues. If the reactive-SH group is involved in biological activity of the protein, the displace- ment of the hydrogen and the addition of a large metal atom to the S atom usually cause a major change in protein structure and loss of function. Hence, heavy metals are often poisons.

In contrast, amino acid residues in proteins may undergo nonenzymatic chemical reactions that may or may not alter biological activity. An example of this type of reaction is the formation of glycated proteins. The amino groups of proteins combine with carbonyl groups of sugars (glucose) to form labile aldimines (Schiff bases), which are isomerized (Amadori rearrangement) to yield stable ketoamine (fructosamine) products (Figure 2-12). The degree of glycation achieved in a protein is deter- mined by the concentration of sugar in the environment of the protein. In glycated hemoglobin, a Schiff-base adduct forms between the sugar and the N-terminal group of the /~-chains of hemoglobin.

The Amadori sugar-amino acid residue adducts in proteins are produced with prolonged hyperglycemia and undergo progressive nonenzymatic reactions involving dehydration, condensation, and cyclization. These compounds are collectively known as advanced glycosylation end products and are involved in the chronic complica- tions of diabetes mellitus (cataracts and nephropathy) (Chapter 22).

Supplemental Readings and References 33

~2COH

.of~ Glucose

-H20 1[

O

H

OH

RNH2 Amino group of a protein

H2COH H2COH OH

HR H -_ ~ p N R

H( H H H /

1 , ,

OH OH Glucosylamine Schiff base

H+[ [ Amadori rearrangement

H2COH

H2R H 2 c ~ O " ~ OH [02]

H( H ' ~ H~//(~H2NHR

OH OH 1 -Amino- 1 -deoxy- 2-keto compound

F I G U R E 2-12

HN I I ((~H2)4

(?H2)3 CH CH I ~ I ~ H2N COON H2 N COOH

Pentosidine

Nonenzymatic reaction between the glucose and a free amino group of a protein.

Supplemental Readings and References

D. G. Dyer, J. K. Blackledge, S. R. Thorpe, and J. W. Baynes: Formation of pentosidine during nonenzymatic browning of proteins by glucose. Journal of Biological Chemistry 266, 11654 (1991).

M. E. Gurney, E B. Curring, E Zhai, and others: Benefit of vitamin E, riluzole, and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis. Annals of Neurology 39, 147 (1996).

R. G. Hankard, M. W. Haymond, and D. Darmaun: Role of glutamine as a precursor in fasting humans. Diabetes 46, 1535 (1997).

S. J. Kuhl and H. Rosen: Nitric oxide and septic shock. West Journal of Medicine 168, 176 (1998).

L. Lacomblez, G. Bensimon, E N. Leigh, et al.: Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Lancet 347, 1425 (1996).

J. Loscalzo and G. Welch: Nitric oxide and its role in the cardiovascular system. Progress in Cardiovascular Diseases 38, 87 (1995).

R. G. Miller, D. Moore, L. A. Young, et al.: Placebo-controlled trial of gabapentin in patients with amyotrophic lateral sclerosis. Neurology 47, 1383 (1996).

R. Safirstein, L. Andrade, and J. M. Vieira: Acetylcysteine and nephrotoxic effects of radiographic contrast agents--a new use for an old drug. The New England Journal of Medicine 343, 210 (2000).

M. Tepel, M. van der Giet, C. Schwarzeld, and others: Prevention of radiographic-contrast-agent-induced reductions in renal function by acetylcysteine. The New England Journal of Medicine 343, 180 (2OOO).

H. Vlassara: Recent progress on the biological and clinical significance of advanced glycosylation end products. J. of Laboratory and Clinical Medicine, 124, 19 (1994).

T. R. Ziegler, K. Benfele, R. J. Smith, et al.: Safety and metabolic effects of 1-glutamine administration in humans. Journal of Parenteral and Enteral Nutrition 14, 1375 (1990).

Biochemistry Book (Chapter 2 Amino Acids)

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