(Woods) Chem-131 Lec-20 & Proteins 1

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1 (Woods) Chem-131 Lec-20 & Proteins 1 Catalytic proteins, or enzymes: Catalyze the synthesis and utilization of proteins, carbohydrates, lipids, nucleic acids, and almost all other biomolecules. Transport proteins: Protein Functions Bind and carry specific molecules from place to place. Regulatory proteins: Control cellular activity. α Amino Acids Except for glycine (R = H), the α-carbon of all amino acids is a stereocenter and each amino acid exists as a pair of enantiomers. Structural proteins: Give physical shape to structures. Contractile til proteins: Provide cells and organisms with the ability to change shape and move. Protective proteins: Defend against invaders and minimize damage after injury. With very rare exceptions, only the L-α-amino acids exist in the proteins of plants and animals. Amino Acids Mammals require all 20 amino acids for protein synthesis but can synthesize only 10 of them. There are 10 essential amino acids that must be obtained from the diet. Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Lysine, Leucine, Histidine (children), Arginine (children) The amino acids are categorized by their R-groups: Nonpolar neutral Polar neutral Polar acidic Polar basic One amino acid is different from the other 19. Proline: the R-group is bonded to both the α-carbon and to the amino group to form a cyclic structure:

2 (Woods) Chem-131 Lec-20 & Proteins 2 Zwitterion Zwitterion: Containing a -COO - group and an NH 3+ group. The net charge on the molecule is zero. Low ph (H + ): equilibrium is to the left (cation) everything is protonated. High ph (OH ): equilibrium is to the right (anion) everything is deprotonated. Amino acids act as Strong Buffers,, keeping the ph of the solution relatively constant as acids or bases are added. Zwitterion Isoelectric Point (pi): Each amino acid has a ph at which almost all of the molecules are present in the zwitterionic form (0 net charge). Zwitterion Most amino acids have isoelectric points in the range Acidic Amino Acids (ASP, GLU) have isoelectric points of 2.77 and 3.22, respectively. Basic Amino Acids (LYS, ARG, HIS) have isoelectric points of 9.74, 10.76, and 7.59, respectively. At a ph below the isoelectric point, the cation concentration increases (net (+) charge: protonation occurs, excess H + ). At a ph above the isoelectric point, the anion concentration increases (net ( ) charge: deprotonation occurs, excess OH ). At physiological ph, the neutral α-amino acids exist as zwitterions with zero net charge, whereas the acidic and basic amino acids have overall charges of -1 and +1, respectively.

3 (Woods) Chem-131 Lec-20 & Proteins 3 Amino acid residues in the peptide are linked by Peptide Bonds. Peptides are referred to as di-, tri-, tetra-, penta-, hexapeptides, etc amino acid residues, the term polypeptide is used. Dehydration Sythesis reaction between the carboxyl and amino groups of different α-amino acid molecules: Writtenwiththeamino terminal amino acid (N-terminal) on the left and carboxyl-terminal amino acid (C-terminal) on the right. Amino Acid Sequences and Constitutional Isomers Two different dipeptides are possible from the reaction of Gly and Ala: The Peptide Bond & Resonance Peptide bond is usually drawn as a single bond, but actually has considerable double bond character (due to resonance) which prevents free rotation about the bond. Almost all peptide bonds are in the trans configuration which is sterically more stable than the cis configuration. The trans-planar nature of the peptide bond plays an important role in determining three-dimensional structure and function in polypeptides. All peptides have one free α-amino group (N-terminal residue), and one free α-carbonyl group (C-terminal residue).

4 (Woods) Chem-131 Lec-20 & Proteins 4 Ionization of Peptides Isoelectric ph: where each peptide is electrically neutral. The pi value of a peptide containing only neutral α-amino acid residues or equal numbers of acidic and basic residues or both is in the range of pi values for neutral α-amino acids (ph ). Electrophoresis A strong electric field causes anions (acidic amino acids) to move towards the anode (positive electrode) and cations (basic amino acids) to move towards the cathode (negative electrode). Amino acids whose isoelectric ph is close to the ph of the solution remain stationary in the electric field. Acidic pi: (lower than ) if there is an excess of acidic residues in the peptide. Basic pi: (higher than ) if there is an excess of basic residues in the peptide. All amino and carboxyl groups are charged at physiological ph, including those on side groups of acidic and basic residues. Peptides have solubility and electrophoresis properties that are ph dependent. Peptide solubility is lowest at its isoelectric point. Reactions of Peptides Cysteine contains a sulfhydryl (thiol) group, -SH. Pairs of cysteine residues often link two peptide chains or two parts of one peptide chain through Disulfide Bridges: 3D Shape of Proteins The formation of a protein s 3-D structure by conformational changes within its amino acid residues is called protein folding. The overall folding of a protein is described at four levels: Primary structure is the α-amino acid sequence of a polypeptide. Secondary structure is the conformation in a local region of a polypeptide molecule. The conformations are the same in different regions of the molecule for some polypeptides but are different in different regions for other polypeptides. Tertiary structure exists when the polypeptide has different secondary structures in different local regions. Tertiary structure describes the three-dimensional relation among the different secondary structures in different regions. Quaternary structure exists only in proteins in which two or more polypeptide molecules aggregate together. It describes the threedimensional relation among the different polypeptides.

5 (Woods) Chem-131 Lec-20 & Proteins 5 Secondary Structure of Proteins α-helix: The polypeptide p chain is arranged like a coiled spring with a hydrogen bond between each peptide group s C=O oxygen and the hydrogen of the N-H group of the fourth residue farther down the chain. α-helix can better accommodate larger side groups than β-pleated sheets (except consecutive large or like charged residues). Secondary Structure of Proteins Tertiary (3D) Structure of Proteins β-pleated sheet: Peptide chains are extended and run side-by-side each other in either a parallel or an antiparallel arrangement. Neighboring chains are held together by hydrogen bonds between an N-H on one chain and a C=O on a neighboring chain. Side chains extend alternately above and below the plain of the sheet.

6 (Woods) Chem-131 Lec-20 & Proteins 6 Normal vs Mutant Prion (Mad Cow Disease) Hemoglobin: A Protein with Quaternary Structure The Structure of a Transmembrane Protein Environmental Factors Affecting Enzyme Activity: Temperature, Salt Concentration, & ph.

7 (Woods) Chem-131 Lec-20 & Proteins 7 Agents that Causes Denaturation Heat: Weak side-chain attractions in globular proteins are easily broken by heat. Mechanical agitation: Denaturation of protein by mechanical agitation is the foaming during beating of egg whites. Detergents: Low concentration can disrupt hydrophobic side chains. Organic compounds: Polar solvents such as acetone or ethanol can interfere with hydrogen bonding by competing for bonding sites. ph change: Excess H + or OH - ions reacts with the basic or acidic side chains in amino acid residues and disrupt salt bridges. Inorganic salts Sufficiently high concentrations of ions can disrupt salt bridges. Heavy metal salts Fibrous Proteins Fibrous Proteins: elongated, water insoluble, proteins that serve structural and contractile functions. Minimal Tertiary Structure: generally a single conformational pattern throughout all or most of the chain (secondary structure). Quaternary: usually an aggregation of two or more polypeptide chains into a specific conformational pattern. α-keratins: structural component of hair, horn, hoofs, nails, skin, and wool. Coiling at higher and higher levels is a mechanism for enhancing strength. Fibrous Proteins: α-keratins Fibrous Proteins: α-keratins Permanent waving of hair (a Perm ) is accomplished by breaking and reforming cysteine cross-links within the hair fiber: The packing within the α-keratins is stabilized by disulfide bridges and secondary forces between different polypeptide molecules. Interchain disulfide bonds are often called cross-links. The degree of hardness of an α-keratin depends upon its degree of cross-linking. High cysteine content results in increased hardness (hair, horn, nail) compared to low cysteine content (skin, callus).

8 (Woods) Chem-131 Lec-20 & Proteins 8 β-keratins and Silk Fibroins Collagen The β-keratins make up the proteins in bird feathers, reptile scales, and silk fibroin (below). β-keratins are almost completely composed of β-pleated sheets. The most abundant protein in vertebrates is collagen. Stress-bearing component of connective tissues such as bone, cartilage, cornea, ligament, teeth, tendons, and the fibrous matrices of skin and blood vessels. Contains much more Glycine/Proline, much less Cysteine than does α-keratin. A collagen fibril is very strong and undergoes little extension when stretched. Collagen A single collagen molecule forms a left-handed helical structure, much more elongated than an α-helix. Three left-handed collagen helices twist around each other. In bone and teeth, collagen fibrils are embedded in hydroxylapatite, Ca 5 (PO 4 ) 3 OH, which is an inorganic calcium phosphate polymer, to form a high physical strength structure. Calcium fluorapatite Ca 5 [(PO 4 ) 3 F], which is more chemically stable and dissolves at a ph of 4.5, compared to 5.5 ph for calcium hydroxyapatite. Leads to fewer cavities, since stronger acids are needed to attack the tooth enamel. Globular Proteins Globular proteins do not aggregate into macroscopic structures but remain soluble in order to carry out their metabolic functions: catalysis, transport, regulation, and protection. Globular proteins remain soluble by folding up in certain patterns: Hydrophobic amino acid side chains in the interior of the molecule, Hydrophilic amino acid side chains on the exterior of the molecule, in contact with water. Lysozyme: an enzyme found in the cells and secretions of vertebrates. Lysozyme: hydrolyzes bacterial cell walls.

9 (Woods) Chem-131 Lec-20 & Proteins 9 Globular Proteins Myoglobin consists of a single chain containing 153 amino acid residues, organized into 8 α-helical regions that surround a heme: Globular Proteins Hemoglobin has 2 α-chains and 2 β-chains. Each α and β chain is folded similar to myoglobin and each contains a heme group capable of carrying oxygen. A space at the center can bind a molecule of 2,3- bisphosphoglycerate (BPG) which regulates the affinity of the hemoglobin molecule for oxygen. The iron atom on the heme group is the site of attachment of the O 2 molecule. Myoglobin has a higher affinity for oxygen than does hemoglobin and serves as a storage reserve for oxygen within the muscle. Carbon monoxide binds more strongly than oxygen to the heme groups in hemoglobin and can result in death from asphyxiation. Heavy smokers tie up a significant fraction of their hemoglobin with carbon monoxide, resulting in shortness of breath. Genetic Mutation: Sickle-cell Anemia & Malaria Sickle-cell hemoglobin is one of the most serious mutations involving hemoglobin. The sickle-cell mutation involves the change of one amino acid on the β-chain: a glutamic acid is replaced by a valine. Deoxygenated red cells containing sickle-cell hemoglobin assume an elongated shape, clogging the capillaries which h become inflamed and cause considerable pain. In deoxyhemoglobin, a hydrophobic pocket is formed into which the hydrophobic valine side chain on a neighboring hemoglobin molecule can fit, leading to polymerization. Eukaryotic Cell

10 (Woods) Chem-131 Lec-20 & Proteins 10 Cellular Metabolism The Complexity of Metabolism Catabolism: Biochemical degradation of energy containing compounds. Conversion of energy simpler molecules which are then used in biosynthesis of cellular components. Anabolism: Biochemical synthesis of biomolecules from simpler components. Ion and biological transport across membranes. Energy Carriers Adenosine triphosphate (ATP): Carrier of energy. Nicotinamide adenine dinucleotide (NADH): Carrier of reducing power to ETS. Nicotinamide adenine dinucleotide phosphate (NADPH): Carrier of reducing power for biosynthesis. Cellular Metabolism: Catabolism ADP - ATP 1. Nutrient molecules are degraded. 2. Converted into acetyl-s-coenzyme A (acetyl-s-coa). 3. Acetyl-S-CoA oxidized to CO 2 and H 2 O. 4. TCA & ETS. Substrate level phosphorylation. Oxidative phosphorylation.

11 (Woods) Chem-131 Lec-20 & Proteins 11 How ATP Powers Cellular Work NAD + - NADH ATP Chemical work Mechanical work Transport work Membrane protein Solute P + Reactants Motor protein P P Dehydrogenation / Hydrogenation Removal-Addition of hydrogen & electrons NAD + is reduced by accepting a hydride ion (H: - ) to form NADH. NADH is reoxidized by passing electrons to oxygen through the ETS. NADPH is a source of reducing power during anabolic biosynthesis. P P Product P Molecule formed Protein moved Solute transported ADP+ P FAD - FMN FAD and FMN: Tightly bound coenzymes to flavoproteins. Also involved in enzymatic dehydrogenations. Energy Profile of an Exergonic Reaction

12 (Woods) Chem-131 Lec-20 & Proteins 12 Energy of Activation (E A ) The Catalytic Cycle of an Enzyme E A without enzyme Energy Reactants E A with enzyme Net change in energy Products Progress of the reaction Environmental Factors Affecting Enzyme Activity: Temperature, Salt Concentration, & ph.

13 (Woods) Chem-131 Lec-20 & Proteins 13 Most enzymes are combined with specialized small molecules called cofactors or coenzymes. The purpose of a cofactor is to maintain the protein portion of the enzyme in the correct conformation. Many catalytic functions cannot be accomplished using the functional groups provided by the amino acid side chains alone. Small organic molecules called coenzymes, carrying the necessary functional groups, are transiently bound to the enzyme: Positive Regulator: changes the activity site so that the enzyme becomes a better catalyst and rate accelerates. Competitive Inhibition: The inhibitor binds to the active site. The binding is reversible and the inhibitor can be displaced by raising the substrate concentration. Noncompetitive Inhibition: The inhibitor binds elsewhere - not to the active site. Raising the substrate concentration does not reverse this type of inhibition. Negative Regulator: changes the activity site so that the enzyme becomes less effective and rate slows down.

14 (Woods) Chem-131 Lec-20 & Proteins 14 Feedback Inhibition The rate of an enzyme catalyzed reaction reaches a maximum value as the substrate concentration is increased. At this point the enzyme is said to be saturated. When saturated with substrate, the enzyme activity is described as the turnover number of the enzyme: the number of substrate molecules converted into product molecules per enzyme molecule per unit time. Enzymes, such as carbonic acid, increase the rate of the catalyzed reaction by a factor of 1 x 10 7 over the uncatalyzed reaction. The rates of enzyme catalyzed reactions with or without a competitive inhibitor.