1014NSC Fundamentals of Biochemistry Semester Summary

Griffith University, Nathan Campus

Semester 1, 2014

Topics include: - Water & pH - Protein Diversity - Nucleic Acids - DNA Replication - Transcription - Translation - Protein Structure & Function - Enzymes - Enzyme Kinetics - Enzyme Inhibitors - Carbohydrates - Lipids - Membranes - Membrane Transport

Module 1.1- Water and pH

- Water plays a central role in the chemistry of all life - Proteins, polysaccharides, nucleic acids and membranes all

assume their characteristic shapes in response to water - Chemical properties of water are related to the functions of

biomolecules, entire cells and organisms

The structure of Water Important properties arise from its angled shape:

- Angle of 104.5o between two covalent bonds o Bonding orbital- sp3

- Polar O-H bonds due to electronegativity of oxygen o Oxygen nucleus attracts electron more strongly o Electrons are more often in the vicinity of the oxygen atom

(2δ-) than hydrogen (δ+) - Angled arrangement of polar bonds creates permanent

dipole o Negative O dipole is equal in magnitude to the two

positive H dipoles

Weak interactions in aqueous solutions Hydrogen Bonding Between water molecules Due to their polarity, water molecules attract each other

- Hydrogen bond formed between positive H dipole and negative O dipole of two water molecules o Relatively weak- bond dissociation energy of 23 kJ/mol (470kJ/mol for O-H covalent bond)

- Water molecule can form up to 4 H-bonds For liquid water

- Molecules are disordered and in constant motion - Each forms H-bonds with average of 3.4 other water molecules

For ice - Molecules form maximum of 4 H-bonds

o Creates regular lattice (uniform structure) o Makes ice less dense than liquid water

Between water and polar solutes Hydrogen bonds are not unique to water

- Form between any hydrogen bond donor and hydrogen bond acceptor o H-bond donor= electronegative atom covalently bound to hydrogen o H-bone acceptor= electronegative atom (usually oxygen or nitrogen)

- H-bonds strongest when molecules are orientated to maximise electrostatic attraction o Straight line so that acceptor atom is in line with covalent bond between donor atom and H

Examples of biologically important hydrogen bonds

Polar, nonpolar and amphipathic molecules Water is a polar solvent (only forms H-bonds with polar molecules)

- Hydrophilic- generally charged or polar, readily dissolved - Hydrophobic- non-polar, poor solvents

Ionic Interactions Water dissolves crystalline salts by hydrating and stabilizing component ions For example NaCl:

- NaCl crystal lattice is disrupted as water molecules cluster about the Cl- and Na+ ions o Ionic charges of ions partially weakened electrostatic attractions between ions reduces

- Ions acquire greater freedom of motion- increase in entropy (favourable free-energy change)

Hydrophobic Interactions Nonpolar gases Biologically important gases (CO2, O2, N2) are nonpolar

- Hydrophobic and poorly soluble in water - Change from disordered gas phase into aqueous solution reduces motion

o Decrease in entropy (energetically unfavourable) Hydrophilic and are poorly soluble in water

o Transitioning from disordered gas phase into aqueous solution reduces motion Decrease in entropy (energetically unfavourable)

Nonpolar compounds Nonpolar compounds interfere with H-bonding among water molecules

- Water molecules in immediate vicinity of nonpolar solute are constrained in possible orientation o Solute becomes surrounded by highly ordered water molecules → decrease in entropy

For amphipathic compounds: - Polar, hydrophilic region interacts favourably with water - Nonpolar, hydrophilic region tends to avoid contact

o Will form micelles (held together with hydrophobic interactions) Disruption of ordered water molecules is driving force in formation of polar enzyme-substrate complexes

o Causes entropy increase as water molecules can become more disordered

van der Waals Interactions Occur between two uncharged atoms in close proximity

- Surrounding electron clouds influence each other - Random variations in electron position creates transient electric dipole

o Induces transient, opposite electric dipole in nearby atom - Weak electrostatic attraction brings two atoms closer

Ionization of water, weak acids, and weak bases Pure water is slightly ionized

- Ionization expressed by an equilibrium constant (Kw)

𝑲 = [𝑯+][𝑶𝑯−]

[𝑯𝟐𝑶]=

𝒑𝒓𝒐𝒅𝒖𝒄𝒕𝒔

𝒓𝒆𝒂𝒄𝒕𝒂𝒏𝒕

- pH scale designates H+ and OH- concentrations

pH = - log10[H+] - Weak acids have characteristic dissociation constants

o Titration curves reveal pKa of weak acids Water is a neutral molecule with a very slight tendance to ionize

- H2O ↔ H+ + OH-

- Free protons (H+) don’t actually exist, rather they exist as hydronium ions, H3O+ o For simplicity- often represented as H+

Proton movement between water molecules Proton of a hydronium ion is highly mobile (“proton hopping”)

- H+ and OH- ions therefore much more mobile than other ions o Reason why acid/base reactions are so fast

Acids and bases Acid = proton donors

- Once acid has lost proton, it becomes the conjugate base Base = proton acceptor

- Once base has accepted proton, it becomes the conjugate acid Strong acids and bases completely dissociate

HA → H+ + A-

Weak acids and bases partially associate as described by their dissociation constant (Ka) - The larger Ka, the stronger the acid - The smaller pKa, the stronger the acid

HA ↔ H+ + A-

Titration Curves Titration is used to determine amount of acid in solution

- Strong base is added until solution is neutralised (pH7) Plot of pH against amount of base added (titration curve) reveals pKa

- At midpoint, half of original acid has dissociated o At this point concentration of acid is equal to

concentration of conjugate base pH=pKa

Stronger acids have midpoint at low pH whereas weaker acids have theirs at high pH

Buffering against pH changes in Biological Systems Buffers are mixtures of weak acids and their conjugate bases

- Buffer cells and tissues against changes in pH - Concentrations of acid and conjugate base should be equal such that pH=Pka - Tend to resist changes in pH where small amounts of acid/base are added

o Result of the acid-conjugate base equilibrium shifting as described by Le Chatelier's principle Decreasing pH causes an increase in acid Increasing pH causes a decrease in conjugate base

o Buffer region ~pKa +/- 1

Henderson-Hasselbalch equation relates pH, pKa and buffer concentration

𝒑𝑯 = 𝒑𝑲𝒂 + 𝐥𝐨𝐠[𝑨−]

[𝑯𝑨]

Three important points

1. pH depends on pKa and [𝐴−]

[𝐻𝐴]

2. [𝐴−]

[𝐻𝐴] depends on pH and pKa

o log[𝐴−]

[𝐻𝐴]= 𝑝𝐻 − 𝑝𝐾𝑎

3. At half-equivalence (midpoint) pH = pKa

o [acid]=[conjugate base]

log[𝐴−]

[𝐻𝐴] = 0

Module 1.2- Protein Diversity Define protein: a macromolecule composed of one or more polypeptide chains, each with a characteristic sequence of amino acids linked by peptide bonds

Proteins - Most abundant biological molecules occurring in all cells and all parts of cells - Range in size from relatively small peptides to huge polymers of molecular weights in the millions - Occur in great variety and exhibit enormous diversity in biological function

o Enzymes o Transport proteins o Nutrient & storage proteins o Contractile & motile proteins o Structural proteins o Defence proteins o Regulatory proteins o Specialized proteins

Amino acids 20 amino acids linked through peptide bonds give rise to the entire array of proteins

- Amino acid= α-amino-substituted carboxylic acids - Peptide bond= a substituted amide linkage between the α-amino

group of one amino acid and the α-carboxylic group of another All amino acids have a carboxyl and amino group bonded to a α-carbon

- The α-carbon also has a hydrogen and a side chain (R-group) - Each amino acid differs in the side chain which vary in charge, size,

structure, water solubility and chemical properties

Chirality of amino acids Amino acids consist of four substituents around a α-carbon, thus they have two possible stereoisomers

- Since they are non-superimposable they are enantiomers Formation of stable repeating structures requires constituent amino acids to the same enantiomer

- Exclusively L isomers are synthesised due to the asymmetric binding sites of enzymes

Nonpolar, aliphatic

- Nonpolar and hydrophobic - Become more hydrophobic as side chain length increases - Usually cluster together within proteins, avoiding aqueous environments

o Stabilizing structure by means of hydrophobic interactions

Proline - Cyclic amino acid - Shares many properties of aliphatic (straight

chain) amino acids - Rigid bond-angles of proline make it difficult to

fold into proteins - Generally introduces a kink into polypeptide

chain

Polar, uncharged amino acids - More soluble in water and hydrophilic - Polarity due to:

o Hydroxyl groups of serine and threonine o Sulfhydryl group of cysteine o Amide groups of asparagine and glutamine

Asparagine and glutamine are the amide derivatives of aspartate and glutamate, respectively

Cysteine - Side chain can ionize at moderately high pH - Can from a covalently linked dimeric amino acid (cysteine)

o Two cysteine residues joined by disulphide bond - Plays special role in structures by covalently linking

different parts of a protein/polypeptide - Disulphide linkages are strongly hydrophobic (nonpolar)

Positively charged (basic) amino acids

Lysine and Arginine - R groups carry positive charges and are strongly polar at pH7 - Very hydrophilic, usually found on exterior of proteins - Lysine- secondary amino group on aliphatic side chain - Arginine- positively charged guanidino group

Histidine - Contains a imidazole group - Only common amino acid ionisable with a pKa near neutrality - Residues facilitate many enzyme-catalysed reactions by serving as a proton donor/acceptor

Negatively charged (acidic) amino acids - R groups carry a net negative charge at pH7 - Very hydrophilic

Aspartate and glutamate have a second carboxylic acid group

Aromatic amino acids - R groups are relatively nonpolar and participate in hydrophobic interactions - Tyrosine and tryptophan are significantly more polar than phenylalanine due to tyrosine hydroxyl

group and tryptophan indole ring - Able to absorb ultraviolet light- allows us to study proteins

.

Modified amino acids There are a number of modified amino acids which are formed from the common amino acids Examples:

Selenocysteine Introduced during protein synthesis rather than created through a post-synthetic modification

- Cysteine but with sulphur replaced by selenium

Other properties of Amino Acids Amino acids are ionised in aqueous solution.

- pKa for substituents will vary

For amino acids without ionisable side chains, at pH 7 zwitterions are formed (dipolar ion)

- Amino group is largely protonated (NH3+) - Carboxyl group is largely deprotonated (COO-)

Amino Acid titration curves Distinct stages correspond to the deprotonation of carboxyl and amide groups and any ionisable side chain

- At these stages regions of buffering power exist Isoelectric point (pI)= pH at which molecule has net charge of zero

- Average of pKa values pH < pI amino acid has net positive charge pH > pI amino acid has net negative charge The side chains of some are amino acids are also ionisable:

- pH > pKa the proton tends to be off - pH < pKa the proton tends to be on

Peptide bond Formed when two amino acids are covalently linked between an amino group and a carboxyl group

Properties of peptide bonds - Planar and prefer to be in the trans form - Have an unreacted amino group at one terminus (amino N terminus) and an unreacted carboxyl group

at the other terminus (carboxyl C terminus)

Module 2.1- Nucleic Acids Four Functions of Genetic Material

1. Must contain complex information o Capable of storing instructions for the traits/functions of an organism

2. Must replicate faithfully o Capacity to be copied accurately and transmitted to descendant cells

3. Must encode the phenotype o Genotype must have capacity to be expressed/code for traits (phenotype)

4. Must have the capacity to vary o Different species/individuals of a species differ in their genetic make up

Nucleic Acids

Primary structure String of nucleotides joined together by phosphodiester linkages

Nucleotides

Sugar Pentose sugar containing 5 carbons, numbered 1’, 2’, 3’… RNA- sugar is called ribose

- Has a hydroxyl group (-OH) attached to the 2’-carbon - Additional oxygen makes RNA nucleotide more reactive/

less chemical stable DNA- sugar is called deoxyribose

- Has hydrogen atom (-H) attached to the 2’-carbon - More stable, therefore better suited as long-term repository of

genetic information

Nitrogenous base Purines- six-member ring attached to a five-member ring

- Adenine (A) and guanine (G) Pyrimidines- six-membered ring only

- Cytosine (C), thymine (T) and uracil (U) o Cytosine present in both DNA and RNA however, thymine

only present in DNA and uracil only present in RNA Nucleoside= deoxyribose/ribose sugar and a nitrogenous base Chargaff’s rule- for DNA, A=T and G=C

Phosphate group - Phosphorus atom bonded to four oxygen atoms - Frequently carry a negative charge which makes DNA acidic

Polynucleotide strands Phosphodiester linkages= strong covalent bonds connecting nucleotides

- Join the 5’-phosphate group of one nucleotide to the 3’-hydroxyl group of the next nucleotide

- Backbone consists of alternative sugars and phosphates Polynucleotide stands have direction/polarity

5’ end- free phosphate group attached to the 5’-carbon 3’ end- free hydroxyl group attached to the 3’-carbs