biochemistry

exam 1

irritability

the capacity of cells to sense their environment, to learn from the world around them, and to adapt by recalling and implementing effective “coping” strategies. Also includes the ability to communicate both within and among organisms.

Motility

the capacity to generate the molecular-scale forces that are needed for cells and organisms to move, for metabolites to be transported across membranes, for sub cellular components to be translocated, for chromatin to be dynamically restructured, for muscle contraction, cell crawling etc.

Reproduction

the capacity to generate entire organisms, thereby renewing their vitality, while correcting any errors.

Sources of redox energy

solar energy: Earth receives 4 x 10^24 Joules/year of sunlight

Geochemical: mainly oxidation of sulfur and minerals spewed from volcanoes & thermal vents; pre-dated photosynthesis

Sources of thermal energy

Sun’s infrared light

radioactivity: 40-50% of Earth’s surface heat comes from nuclear fission deep within its core

Steady-state balance: rate of heating = rate of cooling- important to create temperate environment that’s conducive for life

Gravitational energy

Earth’s rotation- day/night cycles and tides

Its near-circular orbit- near-constant temperature and moderate seasons

Earth’s tilt- attenuation of damaging UV light striking Earth’s surface

Capturing Solar Energy: photosynthesis

Life thrived, once there were pigments capable absorbing the suns considerable output of electromagnetic radiation

NADH- Nature’s hydride battery

by accepting electrons as hydride ions, NAD+ becomes NADH, thereby allowing cells to store/manage chemical energy

Hydride anion

a hydrogen atom with one more electon

H-

Draw NADH/NAD+ oxidation/reduction

Oxidative Phosphorylation

NADH oxidation supplies energy to make 3 mol ATP from 3 mol ADP & 3 mol phosphate

bioenergetics- biological thermodynamics

defined as the systematic study of energy-transducing processes in cells, with the goal of learning:

how cells extract energy from their environment

how they use this energy to synthesize biomolecules and/or drive energy-requiring processes

how the efficiency of biological pathways changes in disease or metabolic stress

Biology obeys the laws of thermodynamics- NO EXCEPTIONS

1st law- energy may change form or may be transported, BUT energy is not created or destroyed

2nd law- the entropy of the universe always increases

equilibrium thermodynamics is path-independent

Gibbs Energy

measure of the work that a system can perform at constant temperature and pressure

key gibbs free equations \*\*\*\*

*delta****G*****°** = delta*H* - *TdeltaS*

delta***G*****°**= -RTlnKeq

DeltaH**°**

standard enthalpy change- measures the standard state change in the internal energy content, as reflected in the number and stability of all bonds and bonding interactions in the products versus reactants

DeltaS**°**

Standard entropy change- measures the standard change in the disorder of a chemical reaction

Enthalpy

total energy of a reacting system plus any available “PV” work: deltaH= H(products)- H(reactants)

DeltaH>0

heat content of the covalent & non-covalent bonds in the products is MORE than the heat content of covalent & non-covalent bonds of the reactants

Heat is absorbed to form products

DeltaH= 0

heat content of the covalent & non-covalent in the products is same as the heat content of the covalent & non-covalent bonds of the reactants

No heat is liberated or absorbed, upon the formation of products

DeltaH

we say that the heat content of the covalent & non-covalent bonds in the products is less than the heat content of the covalent & non-covalent bonds of the reactants

Heat is liberated when products form

Entropy

represents the fraction of a system’s energy that, as a result its randomness, cannot be converted into useful work

TdeltaS = T (S(products)- S(reactants))

DeltaS>0

there is more disorder in the products than the reactants

overall randomness increases as products form

DeltaS=0

overall disorder does not change when products form

overall randomness is unchanged as products form

DeltaS

overall disorder of the products is less than the overall disorder of those of the reactants

overall randomness decreases as products form

Standard Free Energy Change, Delta***G*****° ******

Reacting systems always move towards equilibrium

at equilibrium, forward and reverse reaction rates are equal

concentrations of reactants and products at equilibrium define the reaction’s equilibrium constant Keq

How Keq and DeltaG**°** are related

DeltaG**°** is a measure of how far a reaction must go to reach its final or equilibrium position

each chemical reaction has its own DeltaG**°** value

Biochemical Standard State Conditions

In chemistry Keq and DeltaG**°** are thermodynamic parameters that apply to chemical standard state conditions

Such conditions are not convenient for studying biochemical processes

Renormalized parameters where Keq’ and DeltaG**°**'‘ (Molarity, ph=7, incorporate H2O concentration and T=37 degrees celsius, P=1 atm and 1-2mM free MG2+)

always use these and indicated by (‘)

The actual free energy change (DeltaG) is the thermodynamic driver within cells (No little circle)

the free energy change for a reaction depends on the actual concentrations of substrates and products, as well as the temperature

Intracellular concentrations are hardly constant, they depend on diet, exercise, and even disease

Delta G uses the actual concentrations

if DeltaG

Key differences between DeltaG and DeltaG**°’ *****

How we measure metabolites in situ?

Wollenberger Tongs

Consists of 2 aluminum blocks, that are joined by a hinge and a long arm with finger rings

They are brought to a very cold temperature

Then without interrupting blood flow and organ/tissue is squeezed between the plates,

The very low temp, the high rate of heat transfer to the metal plates results in freezing very fast

can analyze samples by many techniques

Isolated system

no energy or mass is exchanged between system and surroundings

closed system

energy is exchanged, but mass is not

open system

both mass and energy are exchanged with the surroundings

Hydrogen bond

stable interaction occurring when two electronegative atoms share a hydrogen bond

H-bonds are constantly made and broken

liquid water is thus a flickering collection of W5 (5 H20 molecules) with extremely little W1

H2O readily forms hydrogen bonds \*\*\*

fundamentally its an acid/base interaction

stronger when all three atoms are aligned

Waters key properties

High heat capacity

High B.P. relative to its molecular weight

high viscosity for its molecular weight

strong interactions with ionic molecules

special interactions with nonpolar molecules

liquid h2o has 3.4 h-bonds on average

ice has 4 h-bonds

H2O deactivates nucleophilic reactions

nucleophilic reactions work best within polar organic solvents, DMF or THF

H2O greatly retards nucleophilic reactions- water crowds electron rich and electron deficient reactants, thereby deactivating them

While seemingly disadvantageous, very beneficial to life

uncatalyzed reactions are extremely slow under physiologic conditions-life requires enzyme catalysis

Glucose phosphorylation

enzymes increase reactivity so metabolism can occur under otherwise unfavorable conditions

catalyzed reactions reaches equilibrium in 2-3 seconds where uncatalyzed reaction takes 1 billion sec

Vital role enzymes play

enzymes bind and dehydrate reactants: increase local concentration of reactants, orient substrates to maximize reactivity, stabilize reaction transition states, make reactions faster

enzymes operate as on/off switches: insignificant reaction occurs in absence of the enzyme, little or no toxic side reactions, highly effective regulation

Acid/base properties of water

undergoes rapid H+ dissociation/reassociation

In reality there is no free H+; instead H+ is transferred to a neighboring H2O, thereby forming hydronium ion H3O+

Proton hopping

in liquid H2O protons hop

autoprotolysis

a proton is transferred between 2 identical molecules; one H2O acts as Bronsted acid (proton donor) and the other H2O molecule acts as Bronsted base (proton acceptor)

pH equations

pH= -log10 \[H3O+\]

Kw= \[H3O+\]\[OH-\]= 10^-14 M^2

pH meters don’t measure \[H+\] but proton activity

pH meters must be standardized with solutions of known concentration and activity, therefore the measured pH of pure water is about 7 not exactly 7

deriving the Henderson-Hasselbalch Equation \*\*\*\*

pH is important in biochemistry

many metabolites have acid or base groups

enzymes have acids and bases on active sites

DNA is polyanion & histones are polycationic porteins

Buffers

weak acids and weak bases that are partially dissociated helping to stabilize pH

buffers obey Le Chateliers principle

stabilize nearly constant pH

stabilize structures of cells, proteins, nucleic acid

biochemists use buffers to stabilize biological structure to assure reproducibility of their experiments

Le Chatelier’s Principle

dynamic systems compensate to an applied stress

Buffer titration curve

buffers are most effective at pH near their pKa where there’s an equally large pool of both weak acid its conjugate base'

effective buffer zone is shown in blue box

pKa is where concentration of conjugate base equals concentration of acid

HCO3-/CO2 buffer system

most important buffer in human acid-base homeostasis

normal arterial plasma pH is 7.4 +/- 0.05 in humans

fluctuations are usually lethal

relative concentrations of HCO3- and CO2 determine plasma pH

uncatalyzed rate is slow and must be accelerated by an enzyme known as carbonic anhydrase

Respiratory compensation

ability of our body to adjust its breathing rate to modify circulating CO2

with pKa of 6.1 would seem that HCO3-/CO2 system would be an ineffective buffer at ph 7.4 however its highly effective because body has respiratory compensation

acidosis

low blood pH, hyperventilation drives CO2 out of the body and reduces \[H+\] returning blood to pH 7.4

alkalosis

high blood pH, rebreathing pushes CO2 into the body, and increases \[H+\] returning blood pH to 7.4

Osmotic pressure of aqueous solutions

minimum pressure that must be applied to a solution to prevent the inward flow of pure water across a semipermeable membrane

measures the tendency of a solution on one side of semipermeable membrane to draw in pure solvent from the other side of a semipermeable membrane

amino acid composition

always have amino group on an alpha carbon (⍺)

stereoisomerism is based on fischer projection for L-glyveradehyde

only alpha amino acids are found in proteins not beta

carboxyl group is on the top and side chain on bottom with hydrogen on right of alpha carbon

Amino acids with nonpolar aliphatic R groups

Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, and Proline

Amino acids with aromatic side-chains

Phenylalanine, Tyrosine, and Tryptophan

absorb UV light

Amino acids with positively charged side-chains

Lysine, Arginine, and Histidine

Have significant side-chain positive charge at pH 7

Histidine is the only basic AA with an ionizable group near neutrality: can serve both as H+ donor or H+ acceptor in enzyme-catalyzed reactions

Amino acids with negatively charged R groups

Aspartate and Glutamate

Amino Acids with uncharged polar side-chains

Serine, Threonine, Cysteine, Asparagine, and Glutamine

Polar R-groups from H-bonds with water

Cysteine & Cystine

Free SH group in cysteine ionizes and is largely undissociated at physiologic pH

Think of cysteine having more electrons, a characteristic of a reduced compound

cystine is mainly present in circulation

Antibody structure is stabilized by S-S bonds

without them- loss of light chain from bloodstream, no antigen recognition and no cross-linking action (agglutination- react with two molecules)

Amino acids are ampholytes

contain both acidic and basic groups and therefore form zwitterion

in between more and less protonated forms

Ionization of amino acids

charge on amino acid depends on solution pH

we begin at low pH (fully protonated) and add OH- (removing acidic protons first)

isoelectric point of amino acids

defined as pH where amino acid has average charge of 0

Peptide bond

one H2O molecule is lost in forming peptide bond

what remains of each AA is called a residue

N-terminus (written on left) and C-terminus (written on right) are unmodified -NH2 and -COOH

peptides formation is by ribosomes

Solid-phase peptide synthesis

Exploits solid-phase resin, on which the peptide is progressively elongated by:

1. loading one amino acid at a time

2. washing and deprotecting to generate newly reactive functional groups

3. chemical coupling to form bond and

4. release of final peptide from resin

Vastness of protein sequence space

20^n

for tetrapeptide 20^4= 160,000

for 400-residues 20^400= 10^520

How to determine isoelectric point of peptides

1. start with fully protonated form

2. remove protons one at a time until you get to +1 form

3. add up the next two pKa values & divide by 2

Determining Protein Sequences

Amino acid sequence is determined in two ways

1. cDNA sequencing

2. Enzymatic/chemical fragmentation, followed by fragment separation and automated sequencing

not good to sequence DNA want to sequence amino acids

have a way to remove a single amino acid at a time-accumulate little bit of each amino acid cleaving and can become very messy

Trypsin cleaves where

carboxyl side of Arg & Lys

Chymotrypsin cleaves where

carboxyl side of Phe (F), Tyr (Y) and Try (W)

How are amino acid sequences written

always with N-terminus on the left, cleavage occurs “AFTER” the specific residue, freeing its COOH group

Cyanogen Bromide (cNBr) Fragmentation

cleaves only after Met to produce two peptides, leaving homoserine lactone lactone at its C-terminus

cleanly fragments protein into a set of peptides

has no effect, if Methionine is on C-terminus

sequencing unknown AA sequence

most proteins have 400-500 amino-acid residues-not possible to chemically sequence entire things so have to cleave protein into smaller fragments

illustrate with 28-residue peptide

1. treat separate samples of pure peptide with Trypsin, Chymotrypsin or CNBr

2. isolate each fragment by chromatography

3. Chemically sequence each fragment

NOTE: we wont know the order of the fragments. So we must use multiple fragmentation methods to obtain sets of overlapping sequences

Edman Sequencing

repetitive removal of AA from N-terminus and subsequent identification

R is different for each amino acid

PTH-AA is identified by gas or liquid chromatography

Process is repeated over and over for 40-50 cycles

automated sequencing of 5-20 ug peptide/protein

Why bother with fragment sequencing?

REMEMBER: often interested in the sequence of the physiologically active form of the protein

Many genes give rise to proteins of different AA sequences, depending on the tissue in which they are expressed

mRNA can be spliced, removing certain sequences and altering the actual sequence of the expressed protein

many proteins are post-translationally modified specialized amino acids

many proteins are post-translationally cleaved, such that the final amino acid sequence does not match sequence encoded in the DNA sequence

best to do both protein and DNA sequencing

Why are noncovalent interactions so vital?

strengths of noncovalent bonding interactions are “tunable”- meaning their weaker energies depend on many factors, such as hydration, electrostatics, polarity etc.

they are made and/or broken readily

they determine protein structure and function; enzyme specificity; DNA structure and function; membrane formation and stability; and nearly every other vital process in living systems

role of weak noncovalent interactions

four basic types

individually weak, the cumulative effect can be very significant

allow proteins and nucleic acids to fold and unfold on a biologically relevant time-scale

H-bonds in Nature

Enzyme-substrate interactions

Receptor-ligand interactions

RNA & DNA base-pairing

H-bonds are easy to make & just as easy to break (fast kinetics)

Role of H-bonding in Proteins

H-bonding is NOT a major driving force in protein folding

formation of extended H-bond networks (especially in the form of ⍺-helix or beta-sheet structures) act as structure-organizing elements

⍺-helix or beta-sheet creates scaffold that helps dehydrate protein’s interior by positioning side-chains to maximize hydrophobic interactions

Electrostatic interactions

attractive and repulsive forces

proteins have numerous ionizable groups

ionic groups tend to be on the surface of proteins

formation of ionic bond releases water

salt bridges

bonds between oppositely charged residues that are sufficiently close to each other to experience mutual electrostatic attraction

salt bridges are a combination of electrostatic interaction and H-bonding

Hydrophobic interactions

strictly speaking they are NOT true bonds

strength of interaction is determined by number of organized water molecules released

also play a dominant role in stabilizing membrane bilayer

Hydrophobic interactions and DeltaG

contrary to intuition, water bind fairly well to apolar groups such that deltaH(water-binding)<< 0 (deltaH(water-release)>>0)

release many water molecules greatly increasing randomness (deltaS(water-release)>>0)

temperature strongly influences deltaG- at lower temperatures deltaG can be unfavorable and at high deltaG can be favorable- it all depends on the value of TdeltaS not just deltaS

Hydrophobicity and protein folding

protein folding decreases local entropy, but H2O release increases entropy- over deltaS is positive

side-chains participating in hydrophobic interactions can swivel, thereby facilitating protein folding

great variety of hydrophobic side-chains maximizes packing and minimized release of bound water

van der Waals interactions

short-lived bond resulting from interactions of fluctuating dipoles on non-bonding electrons

net effect is a weak bond

operate over very short length scale for two spherical electrostatic fields

van der Waals and proteins

water interferes with van der Waals but within protein’s dehydrated interior, numerous van der Waals interactions contribute to protein stability

help hold proteins in their compact shape

Pi-cation interactions

aromatic groups have highly delocalized pi-electron orbitals above and below plane of ring

orbitals often interact with quaternary ammonium ions to form stable bonds that are important in enzyme-substrate interactions and receptor-agonist interactions

in aqueous media, the cation-pi interaction is comparable (potentially stronger than) ammonium-carboxylate salt bridges

aromatic side chains of phenylalanine, tryptophan, tyrosine, histidine are capable of binding to cationic species, such as quaternized amino groups, metal ions, neurotransmitters and pharmaceutical agents

Proteins role in cells

structural elements- collagen, histones, keratins

enzymes- catalysts for metabolism and signaling

antibodies- immune surveillance

receptors & channels- signal transduction

transporters- metabolites import/export

molecular motors- translocation

transcription factors- gene control

protein shape

spherical (preferred term is globular)

Peptide bond characteristics

consensus of bond-lengths and bond angles

C-N peptide bond length is consistently shorter than the C-N bond in most amides

\

Planarity of peptide bond \*\*\*

C-N bond has 40% double-bond character

resonance explains shorter bond-length

C=O and N-H atoms within peptide bond are approximately coplanar (very important)

planarity of multiple AA/peptide bonds

each shaded bond system behaves as planar unit

allows proteins to fold rapidly- reduced freedom greatly facilitates this

amino acid side chains direct the folding of the nascent (newly formed) polypeptide into a functional protein and do much to stabilize that final conformation

torsion angles of peptide bond

three main ones

psi O=C-C(a)

omega O=C-N (peptide bond)

phi N-C(a)

\*look up symbols to make sure you know them

trans-peptide bonds is observed more than cis-peptide bonds

prolyl peptide bonds

cyclic side-chain reduces energetic barrier to adopting cis conformation

most often adopt cis conformation

cannot fit in a=helices, often found at end- helix terminator

secondary structure

helix and sheet structures that arise from the formation of regularly spaced H-bonding between main-chain atoms

Principal secondary structures include:

a-helix

parallel and antiparallel b-pleated sheets

turns- short sequences that connect helices and/or sheets and allow structure that are compact and stable