Amino Acids, Peptides, Proteins and Enzymes Peptides and proteins are encoded by DNA and are built from amino acids

Amino Acids, Peptides, Proteins and Enzymes Amino acids (AA) zwitterionic at pH Proteinogenic amino acids – exist as the L-enantiomers * Spell “CO-R-N” clockwise with H in front Which amino acid is achiral? Which amino acids have two chiral carbons? * Some bacteria use D-amino acids to evade host defense proteases

Physical Properties of Amino Acids Water soluble High melting points Low solubility in organic solvents (Et 2 O) Amino acids are abbreviated with a 3-letter or a 1-letter abbreviation

Amino Acid Side Chains Have Different Properties Non-polar – examples: phenylalanine (Phe, F), valine (Val, V) Polar – examples: serine (Ser, S), Asparagine (Asn, N) Acidic – aspartic acid (Asp, D), Glutamic acid (Glu, E) Basic – Lysine (Lys, K), Arginine (Arg, R)

Peptides and Proteins Differ in the Number of Amino Acid Residues Dipeptide: 2 AA Tripeptide: 3 AA Tetrapeptide: 4 AA Oligopeptide: 2-10 AA Polypeptide: >10 AA Protein: >50 AA AA1AA2AA3AA4 N-terminusC-terminus Amino acid “residues” in peptides and proteins refer atoms remaining after loss of water in condensation reactions to synthesize them.

Peptides and Proteins Differ in the Number of Amino Acid Residues Tetrapeptide Val-Ala-Gly-Phe (VAGP) ValAlaGlyPhe N-terminus C-terminus Biosynthesis: N-terminus to C-terminus Nomenclature: N-terminus to C-terminus Drawn: N-terminus to C-terminus Chemical synthesis: C-terminus to N-terminus

Peptide Bonds: Strong Pi-Donation From Nitrogen to Carbonyl Carbon Peptides drawn in plane of paper with side chains extending forward or backward S-trans preferred over S-cis Peptide bonds do not freely rotate

Peptides and Proteins Are Synthesized In Vivo by Condensation Reactions Amino acid “residues” in peptides and proteins refer to the remaining atoms after the loss of water in the condensation reaction.

Peptides and Proteins Are Degraded In Vivo by Hydrolysis Reactions Hydrolysis of peptides and proteins is catalyzed by proteases (sometimes peptidases or proteinases)

Aspartame Artificial sweetener, dipeptide Glutathione Antioxidant present in body – thiol can be oxidized to disulfide sparing other biomolecules from oxidation Oxytocin (Petocin = name of drug) Peptide hormone that stimulates uterine contractions, lactation Examples of Biologically Active Peptides

Many Hormones Are Peptides or Proteins Hormones are chemical substances secreted by cells or glands that regulate the metabolic functions of other cells in the body. Growth hormone (GH) – Anabolic protein hormone, stimulates bone and muscle growth Antidiuretic hormone (ADH, vasopressin) – Peptide hormone, inhibits urine formation Parathyroid hormone (PTH) – Protein hormone, controls Ca 2+ balance

Insulin stimulates the synthesis of energy storage molecules: glycogen, triglycerides, proteins High blood glucose stimulates insulin secretion Type 1 diabetes mellitus: Insulin secretion is reduced or absent. Type 2 diabetes mellitus: Cells are not responsive to insulin. Lack of insulin activity leads to hyperglycemia (high blood glucose sugar). Insulin is a Peptide Hormone: Stimulates Anabolic Processes

Insulin Peptide Chains Covalently Linked by Disulfide Bridges Amino acid cysteine contains a thiol side chain. Two cysteine residues within a peptide or protein can form a covalent bond - disulfide bridge. Oxidation or reduction of cysteine residues to the disulfide bridge?

Insulin and Other Peptide and Protein Therapeutics Administered via Injection Peptides and protein therapeutics are not orally bioavailable Proteases would degrade insulin via hydrolysis reactions to inactive fragments – smaller peptides and constituent amino acids Other protein and peptide therapeutics: Trastuzamab (Herceptin) Infliximab (Remicade) Human growth hormone (somatropin) Oxytocin (Petocin) Erythropoeitin (EPO)

Protein Structure 1)Primary structure – amide bonds (covalent) 2)Secondary structure – Hydrogen-bonds (non-covalent) 3)Tertiary structure – Hydrogen-bonds Dipole-dipole interactions Hydrophobic interactions Salt bridges Disulfide bridges (non-covalent except disulfides) 4)Quaternary structure – same forces as tertiary structure

Primary Structure of Proteins is the Sequence of Amino Acids Amino acids are covalently linked via amide bonds

Primary Structure of Proteins is the Sequence of Amino Acids With increasing length of peptide or protein chain, an exponential number of amino acid sequences possible Consider tripeptide composed of leucine (L), phenylalanine (F), and alanine (A) How many possible tripeptides can be formed from these three amino acids?

Primary Structure of Proteins is the Sequence of Amino Acids With increasing length of peptide or protein chain, an exponential number of amino acid sequences possible Consider tripeptide composed of leucine (L), phenylalanine (F), and alanine (A) Six possible tripeptides: LFA LAF ALF AFL FLA FAL

Secondary Structures of Proteins Based on Highly Regular Local Sub-Structures Alpha (  )-helices and beta (  )-sheets most common types of secondary structures Based on hydrogen bonding (non-covalent interactions)

Secondary Structure of Proteins: Alpha Helix Spring-like structure formed by hydrogen bonds between the backbone NH and C=O groups approximately 4 amino acids apart Most common type of secondary structure

Antiparallel strands Parallel strands Secondary Structure of Proteins: Beta Sheet Accordian-like structure formed by hydrogen bonds between backbone NH and C=O groups in different parts of the same chain or different polypeptide chains

Tertiary Structure of Proteins – Overall Three Dimensional Shape Next higher level of complexity - folding of the  -helical and/or  -pleated regions H-bonding, dipole-dipole interactions, London dispersion forces, disulfide bridges Tertiary structure of myoglobin, an oxygen-binding protein in muscle Connecting turns/loops Alpha helices Heme prosthetic group (binds O 2 )

Quaternary Structure is Found in Some Proteins: Aggregation of Two or More Polypeptide Chains to Form a Complex Hemoglobin is formed from four polypeptide chains associated with each other primarily through hydrophobic interactions (non-polar residues buried, “escape” water) 2 identical  -chains 2 identical  -chains 4 heme prosthetic groups

Fibrous and Globular Proteins Fibrous proteins (structural proteins) Often only secondary structure Insoluble in water Chemically stable Provide mechanical support and tensile strength to tissues Globular proteins (functional proteins) Compact, spherical proteins with tertiary structure (some quaternary) Water soluble Chemically active

collagen Collagen Most abundant protein in the body. Tensile strength of bones, tendons, ligaments. Keratin Structural protein of hair and nails Elastin Durable and flexible – found in ligaments Fibrous (Structural) Proteins elastin

Globular (Functional) Proteins chymotrypsin Enzymes Protein catalysts Transport proteins Hemoglobin (oxygen), lipoproteins (lipids and cholesterol), protein channels (cell membranes) Metabolic proteins Hormones Defense proteins Antibodies (immunoglobulins), molecular chaperones (aid in protein folding) ion channel protein

Globular Proteins Sensitive to pH and Temperature Changes Denaturation – Loss of three-dimensional protein structure and function Intramolecular hydrogen bonds disrupted at high temperature or pH disturbances Risk of high fever, acidosis, or alkalosis – loss of protein function Chemical denaturation 1)Reducing agents – Cys disulfide to Cys thiols 2)Detergents - disrupt hydrophobic interactions

Enzymes Are Biological Catalysts - Accelerate Reaction Rates Enzymes are generally very substrate specific, and can be stereospecific. Some enzymes are less specific, e.g. alcohol dehydrogenase. E = enzyme S = substrate (reactant) P = product Enzymes accelerate reaction rates by bringing substrates into proper orientation for bond breaking / bond formation

Enzyme Specificity Can Vary Alcohol dehydrogenase catalyzes oxidation of ethanol, methanol, and ethylene glycol Methanol is toxic – 10 mL can cause blindness due to its metabolite formic acid. Treatment may include alcohol dehydrogenase inhibitor (fomepizole, Antizol®) and ethanol Fomepizole and ethanol both compete with methanol for binding alcohol dehydrogenase. Fomepizole (Antizol®)

Enzymes Accelerate Reaction Rates by Lowering Their Activation Energy Enzymes do not alter the equilibrium concentrations of reactant and product (affect kinetics, not thermodynamics)

Enzyme Activity Enzymes operate under specific conditions – pH optimum, temperature optimum Most near pH 7.4, 37 °C, though some differ e.g. pepsin at pH 1.5, found in stomach May require cofactors – ions or organic compounds (“prosthetic groups” if tightly bound) Zn 2+ (alcohol dehydrogenase, metalloproteases) Fe 2+ (peroxidases) NADH, NAD+ NADPH, NADP+ FADH 2, FAD CoA

Many Drugs Are Enzyme Inhibitors Atorvastatin (Lipitor) HMG-CoA reductase inhibitor Lowers LDL cholesterol Celecoxib (Celebrex) COX-2 inhibitor Reduces prostaglandin synthesis (NSAID) Acetazolamide (Diamox) Carbonic anhydrase inhibitor Increases loss of bicarbonate via urine (treatment for alkalosis)

Competitive Enzyme Inhibitors Bind Enzyme Active Site Competitive enzyme inhibitors generally have similar chemical structure as endogenous substrate

Methotrexate (MTX) Inhibits dihydrofolate reductase Anticancer agent (leukemia) Antiinflammatory (rheumatoid arthritis) Folic acid (vitamin B 9 ) Important in pregnancy and infancy Rapidly dividing cells Competitive inhibitor of DHFR Methotrexate is an Example of a Competitive Enzyme Inhibitor

Non-Competitive Inhibitors Bind Allosteric Site on Enzyme Inhibitor SubstrateEnzyme Substrate Enzyme- inhibitor complex Conformational change Non-competitive enzyme inhibitors generally have distinct chemical structure from endogenous substrate.

Acetylcholine Neurotransmitter Substrate for acetylcholinesterase Non-Competitive Inhibitors Bind Allosteric Site on Enzyme Tacrine (Cognex) Alzheimer’s disease Inhibits acetylcholinesterase Non-competitive inhibitor of acetylcholinesterase