Amino Acids, Peptides, Proteins and Enzymes

Peptides and proteins are encoded by DNA and are built from amino acids

DNA RNApeptide

orprotein

transcription translation

Amino Acids, Peptides, Proteins and Enzymes

Amino acids (AA) zwitterionic at

pH 7.4

20 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?

H3NO

O

R

* Some bacteria use D-amino acids to evade host defense proteases

Physical Properties of Amino Acids

H3NO-

O

R

Water soluble

High melting points

Low solubility in organic solvents (Et2O)

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)

OHNH2

O

H3NO

O

R

O

O

O

O

NH3 NH

NH2

NH2

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

NH

HN

NH

OH

O

H2N

R1 O

O

O

R2

R3

R4

AA1 AA2 AA3 AA4

N-terminus C-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)

Val Ala Gly Phe

N-terminus C-terminus

NH

HN

NH

OH

O

H2N

O

O

O

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

NH

HN

NH

O

O

O

NH

O

HN

O

Peptides and Proteins Are Synthesized In Vivo by Condensation Reactions

OH

OHN

R1NH

OHN

R1 O

R2

H2N

O

R2

++ H2O

peptide bond

Amino acid “residues” in peptides and proteins refer to the remaining atoms

after the loss of water in the condensation reaction.

OH

OHN

R1H2N

O

R2

+NH

OHN

R1 O

R2

+ H2O

peptide bond

Peptides and Proteins Are Degraded In Vivo by Hydrolysis Reactions

Hydrolysis of peptides and proteins is catalyzed by proteases

(sometimes peptidases or proteinases)

AspartameArtificial sweetener, dipeptide

GlutathioneAntioxidant 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 Ca2+ 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.

HN

NH

O

NH

HN

O

S

S

NH

HN

O

HS

HN

NH

O

SH

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-bondsDipole-dipole interactionsHydrophobic interactionsSalt bridgesDisulfide 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:

LFALAFALFAFLFLAFAL

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=Ogroups 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 groupsin 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 O2)

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 -chains2 identical -chains

4 heme prosthetic groups

Fibrous and Globular Proteins

Fibrous proteins (structural proteins)

Often only secondary structureInsoluble in waterChemically 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

CollagenMost abundant protein in the body. Tensile strength of bones, tendons, ligaments.

KeratinStructural protein of hair and nails

ElastinDurable and flexible – found in ligaments

Fibrous (Structural) Proteins

elastin

Globular (Functional) Proteins

chymotrypsin

EnzymesProtein catalysts

Transport proteinsHemoglobin (oxygen), lipoproteins (lipids and cholesterol), protein channels (cell membranes)

Metabolic proteinsHormones

Defense proteinsAntibodies (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 + S ES E + P

E = enzymeS = substrate (reactant)P = product

Enzymes accelerate reaction rates by bringing substrates into proper orientation

for bond breaking / bond formation

alcohol dehydrogenase

H

OOH

ethanol acetaldehyde

OH

O

aldehydedehydrogenase

acetic acid

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.

NH

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)

Zn2+ (alcohol dehydrogenase, metalloproteases)

Fe2+ (peroxidases)

NADH, NAD+

NADPH, NADP+

FADH2, 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 B9)Important in pregnancy and infancy

Rapidly dividing cells

N

N N

N

H2N

NH2 N

O

NH

HOOC

HOOC

N

N N

N

H2N

OH NH

O

NH

HOOC

HOOC

Competitive inhibitor of DHFR

Dihydrofolate

Dihydrofolate reductase

TetrahydrofolateFolate

DNA synthesisRNA synthesis

(DHFR)

Methotrexate is an Example of a Competitive Enzyme Inhibitor

Non-Competitive Inhibitors Bind Allosteric Site on Enzyme

Inhibitor

Substrate EnzymeSubstrate

Enzyme-inhibitor complex Conformational

change

Non-competitive enzyme inhibitors generally have distinct chemical structure from endogenous substrate.

ON

O

AcetylcholineNeurotransmitter

Substrate for acetylcholinesterase

ON

O

OH

O

HON

acetylcholinesteraseH2O+ +

Non-Competitive Inhibitors Bind Allosteric Site on Enzyme

N

NH2

Tacrine (Cognex)Alzheimer’s disease

Inhibits acetylcholinesterase

Non-competitive inhibitor of acetylcholinesterase