Amino Acids: Hydrophobic and Hydrophilic Types

Amino Acids

• Amino acids have four functional groups around the α-carbon: α-amino group, α-carboxyl group, a unique side chain (hydrogen in glycine), and hydrogen.
• The α-carbon's asymmetry results in two optically active (chiral) isomers: L- and D-amino acids.
• L-form amino acids are unique to proteins, while D-form amino acids appear in bacterial cell walls and some antibiotics.
• The genetic code in DNA specifies 20 amino acids for polypeptide construction.
• Amino acids are grouped/classified by hydrophobicity and charge properties for understanding their location in proteins and their influence on protein structure.

Hydrophobic and Hydrophilic Amino Acids

• Hydrophobic amino acids have nonpolar side chains, typically found in a protein's interior or where the surface interfaces with lipids.
• Alanine and glycine have the smallest side chains; glycine lacks a side chain, making it compatible with hydrophobic environments.
  • Alanine is prominent in nitrogen transport from muscle to liver during fasting (alanine cycle).
• Valine, leucine, and isoleucine are branched-chain amino acids with altered metabolism in maple syrup urine disease.
• Proline has a cyclized side chain joined back to its α-amino group, forming an "imino" acid that functions as a helix breaker in the secondary structure.
  • Hydroxyproline is also hydroxylated after incorporation into collagen (requires ascorbic acid).
• Phenylalanine, tyrosine, and tryptophan are aromatic amino acids.
  • High serum and tissue phenylalanine levels characterize phenylalanine hydroxylase deficiency (phenylketonuria or PKU).
  • Tyrosine, which cannot be synthesized from phenylalanine in PKU, is a precursor to dopamine and catecholamines and phosphorylated by tyrosine kinases in proteins.
  • Tryptophan serves as a serotonin and melatonin precursor, convertible to niacin.
  • Aromatic amino acids are primary chymotrypsin cleavage sites in proteins.
• Methionine is a sulfur-containing amino acid that is always the first amino acid incorporated into polypeptides, but may be removed afterward.
  • S-adenosyl methionine serves as a single carbon donor.
  • Methionine is the cyanogen bromide cleavage site in proteins.
• Hydrophilic amino acids have side chains that form hydrogen bonds and are found where the surface interfaces with water.
• Serine and threonine are hydroxyl-containing amino acids that can be phosphorylated by various kinases.
  • Serine is a single carbon donor to tetrahydrofolate (THF) to produce N5,N10-methylene THF and glycine.
• Cysteine, a sulfur-containing amino acid, similar to its hydrophobic counterpart methionine, has a thiol group that undergoes enzyme-catalyzed oxidation, but is also sensitive to air oxidation to form cystine.
  • Cysteine is a glutathione component, which is a recyclable antioxidant in cells, and can form covalent disulfide crosslinks that stabilize protein structure, especially secreted proteins.
• Aspartate, asparagine, glutamate, and glutamine are the acidic amino acids and their amides.
  • Aspartate and glutamate carry a negative charge at pH 7.
  • Aspartate is interconverted with oxaloacetate by aspartate aminotransferase (AST).
  • Glutamate is interconverted with α-ketoglutarate by alanine aminotransferase (ALT).
  • Glutamine is formed by glutamine synthetase action in the brain/liver to detoxify ammonia, serves as amide nitrogen donor in purine/pyrimidine biosynthesis.
• Lysine, histidine, and arginine are the basic amino acids that carry a positive charge at pH 7.
  • Lysine and arginine are the site of trypsin cleavage and are present at high concentrations in histones.
  • Histidine is weakly basic, uncharged at pH 7, and forms one of the six coordination bonds with Fe++ in hemoglobin/myoglobin's heme prosthetic group.
  • Arginine (pKa ~14) always has a positive charge at neutral pH and has a crucial role in anionic molecule binding, such as nucleic acids.

Carbohydrates

• Carbohydrates, also known as sugars, can be described as polyhydroxy aldehydes or ketones, and their general molecular formula is Cx(H2O)x, where x = 6 for a hexose.
• Hydroxyl, aldehyde, and ketone groups are potential sites for reaction/modification to produce carbohydrate derivatives.

Carbohydrate Nomenclature

• Carbohydrate length depends on the number of monomers.
• Monosaccharides are simple sugars composed of glucose, fructose, or ribose.
• Disaccharides are composed of 2 monomers, such as lactose, sucrose or maltose.
• Oligosaccharides are composed of 2-10 monomers, such as blood group antigens.
• Polysaccharides are composed of 10+ monomers, such as starch or glycogen.
• If the carbonyl is an aldehyde, the sugar is an aldose. If the carbonyl is a ketone, the sugar is a ketose.
• The prefix denotes the number of carbons (e.g., triose [3C], pentose [5C], and hexose [6C]).

Carbohydrate Structure

• Carbohydrates can exist in open-chain (linear) or cyclized (ring) forms.
• The open-chain form (Fischer projection) has the most oxidized O2 at or near the top.
• At least one carbon is asymmetric, making the molecule optically active (rotates polarized light).
• Carbons are numbered starting at the Fischer projection's top (oxidized end).
• The D- or L- configuration depends on the hydroxyl group position on the carbon furthest from the carbonyl (e.g., if it is on the right, it is a D-sugar).
• An equal mixture of D- and L- forms is called a racemic mixture.
• Sugars differing at only one carbon atom are called epimers (e.g., glucose and galactose).
• D- and L- refer to the configuration around the carbon, not the rotation of polarized light; the terms dextrorotatory and levorotatory refer to rotation of light to the right or the left, respectively.
• Condensation of a hydroxyl with the carbonyl produces a cyclic structure, referred to as a hemiacetal or a hemiketal. The flat, cyclic form (Haworth projection) has the most oxidized O2 at or near the right.
• Deoxyribose and fructose may form five- or six-membered furanose rings
• Glucose exists primarily as a six-membered pyranose ring. Cyclization creates a new asymmetric center at the carbonyl (anomeric) carbon.
• The cyclic form of glucose is in equilibrium with the open-chain form (mutarotation) in a 40,000:1 ratio and creates a racemic mixture of α-anomers (a-hydroxyl pointing down) and β-anomers (a-hydroxyl pointing up).
• Anomers differ only in the configuration at the first anomeric carbon.

Glycosidic Bonds and Polymerization

• Glycosides are formed when the hydroxyl group on a sugar's anomeric carbon and another molecule's hydroxyl group condense to form an acetal or ketal linkage, known as a glycosidic bond.
• Glycosides formed from glucose are glucosides; likewise, those from fructose are fructosides.
  • If the second molecule forming the acetal is a sugar, then the glycoside is a disaccharide.
• Glucose polymerization occurs by successive formation of glycosidic bonds between the monomer's anomeric carbon and a hydroxyl group of the growing polysaccharide.
• Like polymerized amino acids and nucleic acids, the linkages in polysaccharides are read from left to right including specification of the anomeric form (e.g., α-1,4 linkages denoting the α-anomer pointing down from carbon 1 of the monomer condensed with carbon 4 of the second sugar).
• Glycosidic bonds stabilize the cyclic form because they prevent formation of the linear structure and mutarotation.
• Reducing sugars are oxidized by Fehling solution to produce color reaction.
  • This includes those sugars whose rings can open to expose the reactive carbonyl groups.
• Reducing sugars include all monosaccharides and oligosaccharides: glucose, galactose, fructose, maltose, and lactose.
• Nonreducing sugars are sucrose and trehalose (ring structures cannot open) and polysaccharides.
  • Large polysaccharides, such as amylose, glycogen, and starch, have one reducing end for each polymer chain, but are generally considered nonreducing polysaccharides.

Disaccharides and Polysaccharides

• Glucose forms glycosidic bonds with itself, fructose, and galactose to produce three nutritionally important disaccharides: sucrose, lactose, and maltose.
  • Sucrose is glucose + fructose.
  • Lactose is glucose + galactose.
  • Maltose is glucose + glucose.
• There are three nutritionally important polysaccharides, all of which are composed entirely of glucose: starch, glycogen, and cellulose.
  • Starch has two major components, amylose and amylopectin.
    • Amylose (α-1,4 linkages) has only a linear structure.
    • Amylopectin (α-1,4 linkages + α-1,6 linkages) has a branched structure; a branch point occurs every 25 to 30 glucose residues.
  • Glycogen is structured like amylopectin, but is exceptionally branched (every 8–12 glucose residues).
  • Cellulose (β-1,4 linkages) has an unbranched structure.
    • Structural polysaccharide of plant cells.
    • Important source of fiber in the diet; not hydrolyzed by digestive enzymes; no caloric value.
• Hyaluronic acid, heparin, and pectin are heteropolysaccharides, since they are formed from several different sugars, including sugar acids and amino sugars.

Carbohydrate Derivatives

• Because of hydroxyl group abundance, sugar molecules can form several types of carbohydrate derivatives.
  • Sugar acids like gluconic and glucoronic acid are produced due to oxidiation of glucose at either carbon 1 or carbon 6.
    • Uronic acids contribute a negative charge to polysaccharide chains which promotes cation binding.
    • Gluconic acid is conjugated with bilirubin in the liver.
    • Ascorbic acid, or vitamin C, is a product of glucuronic acid metabolism, except in primates and guinea pigs.
  • Deoxy sugars are produced when ribose is reduced at carbon 2 to produce 2-deoxyribose.
  • Sugar alcohols, known as polyols, have no carbonyl groups. The aldoses' or ketoses' aldehyde or keto group is reduced, yielding a nonreducing polyol.
  • Amino sugars, produced by replacing the hydroxyl group on carbon 2 by an amino group, creates glucosamine and galactosamine. The amino group is usually acetylated, yielding a neutral sugar.
  • Sugar esters, created when reacting phosphoric acid with one or more hydroxyl groups, produce sugar esters such as glucose 6-phosphate.

Fatty Acids

• Fatty acids are monocarboxylic acids made up of unbranched aliphatic carbon chains.
  • Most (>95%) have an even number of carbon atoms with a chain length of 16 to 20 carbons.
  • Some odd-numbered carbon atom fatty acids are found in the diet.
  • Fatty acid carbons are either saturated with hydrogens or unsaturated when they contain one or more carbon-carbon double bonds.
  • They are classified as short chain (2 to 4 carbons), medium chain (6 to 12 carbons), or long chain (14 to 26 carbons).
• Fatty acids are named by either a common name or a systematic name.
  • Saturated fatty acids are named by their length, and unsaturated fatty acids are named by the position of the double bonds.
• Unsaturated fatty acids have two numbering systems to designate the position of the double bonds: delta numbering system and omega numbering system. Delta numbering system, designated as a three numbering system of number of carbons, number of double bonds, and position of the double bonds - (e.g., linoleic acid has systematic designation of 18:2:Δ9,12 for 18 carbons, two double bonds, double bond after carbons 9 and 12 from the carboxyl end). - Omega numbering system, designated by distance from the most distal (methyl) carbon from the carboxylic acid, which is called the ω carbon (e.g., omega-3 fatty acids have one double bond between the third and fourth carbon from the end of the molecule).
• Carbons 2 and 3 are also referred to as the α- and β-carbons; they are located at positions α and β to the carboxyl group.
• The configuration around unsaturated bonds is designated as cis or trans.
  • Naturally occurring fatty acids always contain cis double bonds, while partially hydrogenated unsaturated fatty acids contain some of the trans form.
• Fatty acids form spherical micelles in water due to their amphipathic properties (possession of both a polar end [carboxylate group] and a nonpolar end [hydrocarbon chain]).
• The melting point of fatty acids is determined by chain length and degree of unsaturation.
  • Increasing length increases melting point.
  • Increasing unsaturation decreases melting point.
  • Cis-unsaturation lowers melting point more than trans-unsaturation.
• The free carboxylate group on fatty acids can present both detergent and osmotic problems in cells that store fats.
• Esterification of the free carboxyl groups with glycerol solves this problem.
  • Glycerol can be esterified with one, two, or three fatty acids to form a monoglyceride, diglyceride, or triglyceride.
  • When fatty acids are esterified to alcohols of 14 to 18 carbons, they are called waxes.
  • Waxes are found in epidermal secretions, such as the outer ear canal.

Nucleic Acids

• Nucleic acids are composed of monomeric units called nucleotides that can join together to form polynucleotides.
• Nucleic acids have several levels of structure.
• As for proteins, primary structure is represented by the linear sequence of nucleotides, and secondary structure involves the regular extended helical structure stabilized by hydrogen bonds.
  • The tertiary structure involves the three-dimensional bending of the helix (discussed further in the chapters on DNA and RNA) as seen in the formation of the compacted tRNA structure or in the formation of nucleosomes by DNA.

Nucleotide Structure

• The nucleotide structure is organized around ribose or deoxyribose
  • A purine or pyrimidine base is attached to the 1'-carbon.
  • One or more phosphate groups are attached to the 5'-carbon.
  • The 3'-carbon is reserved for linkage to the phosphate of another nucleotide during polymerization.
  • The 2'-carbon determines whether the nucleotide is a deoxyribonucleotide (2'-H in place of OH) or a ribonucleotide (2'-OH).
  • DNA and RNA are polymers of deoxyribose and ribose linked by phosphate diester linkages between the 3' and 5' hydroxyl groups of successive pentose units.
• Five bases are found in RNA and DNA
  • Uracil is found in RNA, and its methylated form (thymine) is found in DNA.
  • Cytosine, adenine, and guanine are found in both DNA and RNA.
  • Unusual bases, such as pseudouracil in tRNA and 5-methylcytosine in DNA, are produced by modification after transcription (posttranscriptional modifications).
  • Pseudouracil contributes to the tertiary structure of tRNA. Methylation protects polynucleotides from nuclease digestion.
• Nucleosides are nucleotides without the phosphate, and they are named for bases that comprise them
• The deoxynucleosides are shown in Figure 2-16.
• Both DNA and RNA are polynucleotides that are linked together by phosphodiester bonds between the ribose moiety of the nucleotides.
  • This creates a “ribose-phosphate” backbone and a 5'-end that is phosphorylated and a 3'-hydroxyl end.

Secondary Structure of DNA and RNA

• The union of two complementary strands of DNA-DNA, RNA-RNA, or DNA-RNA occurs through precise complementary pairing of every purine and pyrimidine base.
  • This generates an extended regular structure with the bases paired toward the center and alternating ribose-phosphate bonds toward the edge.
• Like the primary structure of a polypeptide, polynucleotides have a sequence of side chains—in this case, the bases.
  • Polynucleotide structure is always written left to right in the 5' to 3' direction.
  • It is usually depicted as a sequence of bases with or without indicating the phosphates, such as pGpApC or guanyladenylcytosine (GAC). In addition to the hydrogen bonds formed during base-pairing, the DNA helix is stabilized by van der Waals and hydrophobic forces resulting from the stacking of adjacent bases.
• RNA-RNA helices are less stable, however, since the 2'-hydroxyl of ribose does not pack as well as deoxyribose in the double helical structure.
• Pairing is permitted only between adenine/thymine (A-T pairs) and guanine/cytosine (G-C pairs), creating an isomorphic relationship between the strands and maintains DNA strands in opposite (antiparallel) directions. The combination of base pairing produces three forms of DNA: B-form, A-form, and Z-form.
  • B-form DNA is the predominant, natural form, with 10 base pairs per right turn and a periodicity of 34 Å per turn.
  • A-form DNA is produced by dehydrating purified DNA and has 11 base pairs per right turn and a periodicity of 26 Å.
  • Z-form DNA is favored by long stretches of alternating C and G and is called Z-DNA. It contains 12 base pairs per left turn and a periodicity of 57 Å.
• Since the bases pair at an angle, the two grooves in the helix are unequal in size
  • The wider groove is called the major groove, and the narrower groove is called the minor groove. The purine and pyrimidine functional groups extend uniquely into both grooves, including the methyl groups on methylated cytosine and on thymine.
• Enzymes and structural proteins can interact with functional groups in both grooves, permitting them to recognize a sequence without unwinding the helix.
• RNA structure reflects its role in gene expression.
  • It is always produced in a single-strand form, called the sense strand.
    • Any single strand of RNA can fold back on itself to form a hairpin (completely paired) or stem-loop (partially paired) structure if it contains complementary sequences that can base-pair.
  • Note that this maintains the antiparallel nature of base-pairing, and stem loops make up much of the structure of tRNA.

Denaturation of DNA

• Similar to proteins, DNA's structure can be denatured by physical and chemical agents by raising the temperature, both strands separate, or “melt” and disruption of complementary base-pairing and of the hydrophobic stacking forces occurs. If a lowered temperature is gradual, complementary strands renature into a double helix.
• During the initial slow nucleation step, short complementary sequences associate through random diffusion, which is followed by a rapid "zipping" step during which the remainder of the complementary sequences align. If the temperature is decreased too rapidly, the nucleation step is prevented. Denaturation agents include high (nonphysiologic) temperature, which disrupts hydrogen bonds formed during complementary base-pairing and reduces hydrophobic stabilization due to base-stacking and a high pH, which creates a strong negative charge on the phosphodiester groups that produces a charge repulsion between strands.
• Denaturation of DNA is measured through the property of hyperchromicity (i.e., an increase in absorbance of a DNA solution [at 260 nm] on denaturation).
  • If the absorbance is plotted against the increasing temperature, a melting curve is produced
  • The midpoint of the melting curve
• The higher melting point occurs in samples that contain more G-C pairs (higher GC/AT ratio) in the B-form of DNA, since G-C pairs have three hydrogen bonds, whereas A-T pairs have only two.
• Furthermore, a steeper melting curve is more likely from homogeneous samples (identical or similar sequences) of DNA molecules.