Biochemistry Exam 1

Front 1

Condensation

Back 1

Elimination of water (amide bond formation, glycoside bond formation)

Front 2

Hydrolysis

Back 2

is cleavage via water

Front 3

Self-replication

Back 3

One of the most important features of biological system is that it can replicate itself. Template based synthesis is key for the precise replication of DNA, or for the transmitting of information encoded in DNA to RNA or proteins

Front 4

Complementarity

Back 4

allows transfer and correction of information.

Front 5

Eukaryotes
Arachaebacteria

Back 5

Eukaryotic system: animals, plants and fungal. They are only a very small portion of the organisms on earth

Arachaebacteria (archaea): are groups of bacteria that distantly related to other prokaryotes; often “extremophiles”
halobacteria: live in high salt concentration
Methanogens: produce CH4
Thermophiles: live in hot springs

Front 6

Evolution

Back 6

Evolution is not directed toward a particular goal = random changes; those that are non-destructive continue
Evolution requires certain degree of flexibility (bio-diversity = less susceptible to one fungal blight)
Evolutionary is constrained by its past, which means that evolution is a gradual process
Evolution is ongoing.

Front 7

compartmentalization (compartmentation)

Back 7

One of the key features of living organism is that they separate their components from the environments by cell membranes.

Advantages of compartmentation:
Separate the cellular environment from the outside
Enrich the molecules of interest, for example, nutrients
Get rid of toxic molecules by efflux

Front 8

Prokaryotes

Back 8

Prokaryotic cells does not have internal compartments. However, the cytoplasm is not homogenous. The cytoplasm is a very viscous environment so there can still be colocalization of macromolecules and small molecules.

Prokaryotic cells are in general 1 – 10 μm in size. (Human hair = 20-200 um)

Front 9

Eukaryotes

Back 9

Eukaryotic cells are 10X bigger, 10 – 100 μm in size.
The key difference between prokaryotic and eukaryotic cells are that the eukaryotic cells have defined nucleus (separated by nuclear membrane).

Besides the nucleus, eukaryotic cells can also have the following membrane-separtated compartments:
Endoplasmic reticulum: site for the synthesis of many cellular components
Golgi apparatus: protein modification and secretion
Mitochondria: aerobic metabolism (the power plant in eukaryotic cells)
Chloroplast: photosynthesis (plants)
Lysosomes: digestion
Vacuoles: used for storage (plants, some bacteria)
Cytoplasm = cytosol + membrane-bound oraganelles. Organized by cytoskeleton

Prokaryotic and eukaryotic cells evolve to suit their living environments.
Prokaryotic cell grow fast and can tolerate significant environmental fluctuations;
Eukaryotic cells evolved to suit stable environments.

Front 10

Coupled Reactions

Back 10

Reduction: Gain of elections
Oxidation: Loss of electrons


Number of Bonds to O or N
−Number of Bonds to H =
Oxidation # of Carbon


Photosynthesis couples favorable redox reactions to unfavorable reduction of CO2
Catabolism couples favorable redox reactions to unfavorable oxidation to CO2

Front 11

Water

Back 11

Tetrahedral shape if lone pairs are included, or bent.
Polar
Hydrogen bonds determine the structure of solid and liquid water (Structured H-bonds in ice
Dynamic, moving, switching, temporary H-bonds in water (constantly exchanging))
Hδ+ end of one molecule interactions with Oδ- end of another molecule

Front 12

Hydrogen Bonding

Back 12

Van der waals distance is outside edge of electron cloud
Covalent bond is complete overlap
H-bond is partial overlap– makes the interaction stronger

Need:
-a Hydrogen bonded to a very electronegative element (O, F, N)
-an electronegative atom with lone pair

Front 13

Dissolution in water

Back 13

Ionic and polar substances interact with the dipoles in water: solvation, hydration

Front 14

Hydrophobic Effect

Back 14

Spontaneous, Entropy Driven

Water becomes more ordered around non-polar (hydrophobic) = entropy loss ( -ΔS)
Hydrophobic species aggregate to minimize surface area contact to water and decrease entropy loss of water. More H2O disorder = + ΔS
Spontaneous: ΔH – TΔS = ΔG

Front 15

Amphiphiles

Back 15

Amphiphiles are molecules with both polar and nonpolar segments (hydrophilic and hydrophobic)
(Amphipathic)

Ex. Lipids (ions of fatty acids—from soaps, w/cation)

Front 16

Micelle

Back 16

Ball structure with hydrophobic core and hydrophilic shell

Wedge shaped Lipids tend to form micelles

Disrupt these structures with detergents, i.e. sodium dodecylsulfate (SDS)–other amphiphiles –use in lab.
Chaotropes: disrupt structure of water: Urea, Guanadinium HCl

Front 17

Bilayer

Back 17

Two layers with hydrophobic pore sandwiched in between two layers of hydrophilic segments.
--prevents crossing of polar molecules though hydrophobic layer

Cylindrical phospholipids tend to form bilayer

Disrupt these structures with detergents, i.e. sodium dodecylsulfate (SDS)–other amphiphiles –use in lab.
Chaotropes: disrupt structure of water: Urea, Guanadinium HCl

Front 18

Autoionization of Water

Back 18

H2O dissociates into H+ (H30+) and OH-
H+ can travel long distance by proton jumps (similar to the current move in electron conductors)
(move faster than diffusion)

That’s why acid/base reaction is the fastest reaction in aqueous solution.

Front 19

Kw

Back 19

Kw = [H+] [OH-] At 25 0C, Kw = 10-14

So then in pure water at neutral pH the [H+] = [OH-] = 10-7

pH = - log [H+] so
pH = 7; acidic pH < 7.0 , basic, pH > 7.0

Front 20

Acids

Back 20

proton donors

Strong acids: transfer all of their protons to H2O
Complete dissociation
Weak = incomplete dissociation

HA = acid A- = conjugate base

Front 21

Bases

Back 21

proton acceptors

Strong vs Weak

Front 22

Acid Dissociation Constant

Back 22

Weak acids and bases dissociate only partially
Defined by Ka
= tendency to ionize

pKa = - log Ka
Strong acids have “unmeasurably large” Ka’s

*Since strong acids transfer all H+ to H2O, strongest acid in water is H3O+
Strongest base in water is –OH.

Front 23

Henderson-Hasselbalch Equation

Back 23

Ratio of acid to its conjugate base
Determine pKa from pH if know ratio

Front 24

pKa trends

Back 24

BELOW pKa, species is mostly protonated

ABOVE pKa, species is mostly Deprotonated

AT pKa, 50/50 split (1:1 ratio):

Front 25

Titration

Back 25

http://www.youtube.com/watch?v=tvCyrBHJfBk

Front 26

Buffers

Back 26

Buffer = Weak acid + conjugate base

1:10 to 10:1 – range in which buffer can most effectively resist changes in pH (addition of acids or bases)

Rule of Thumb: Effective buffer when pH = pKa ± 1

Front 27

Polyprotic Acids

Back 27

Different pKa for each proton removal.
Can only see discrete steps if pKas are sufficiently separated
At 1st equivalence point, pH = ½ (pKa1+pKa2)

http://www.youtube.com/watch?v=dbddD-qHdTw

Front 28

Protein Building Blocks

Back 28

0 “standard” amino acids are building blocks of proteins
Made up of carboxylic acid and amino group on alpha carbon = “alpha-amino acid”
“R-group” is side chain specific to each amino acid
**Note: Alpha carbon is CHIRAL – has 4 different groups attached

Chiral center = tetrahedral with 4 different groups (as long as R is different)

Front 29

Fischer Projections

Back 29

Rules: carboxyl/carbonyl at top; heteroatoms across from H’s going down (R group at bottom)

Most amino acids are L

Front 30

Zwitterions

Back 30

At physiological pH (~7.4) amino and carboxylic acid groups ionize.
pKa (-COOH) = 3.5
pKa (NH3-) = 9.0

Dipolar ions = zwitterion
Amphoteric- can act as an acid as well as a base

Front 31

Isoelectric Point

Back 31

pI = the pH at which a molecule carries no net electric charge
With only 2 groups, pI = ½ (pK1 + pK2)
Therefore, pI of nonpolar, most uncharged polar is (3.5+9.0)/2 = 6.25

For those with R groups, depends on how charges balance
Acidic = ½ (pK1+pKr)
Basic = ½ (pKr+pK2)
The 2 pKas chosen must be the ionizations that involve the neutral species:

If pH is BELOW pI, then protein is PROTONATED (+ charges or neutral = overall positive charge)
If pH is ABOVE pI, then protein is DEPROTONATED ( - charges or neutral = overall negative charge)

Front 32

Peptide Bond Formation

Back 32

Condensation reaction (elimination of water)

Di, tri, tetrapeptide
Oligopeptide, polypeptide
Amino terminus (N-term)
Carboxy terminus (C-term)

Write N (start) to C (end)

Front 33

4 levels of protein structure

Back 33

primary, secondary, tertiary, and quaternary

Front 34

secondary structure

Back 34

Alpha helix:

α helix is right handed, built from adjacent residues

Each turn has 3.6 residues and span 5.4 Å on average (rise/pitch)

The structure is held together by backbone H-bonds: the C=O bond at n residue H-bonds with the amino group of n+4 residue.

α helix is tightly packed and its atoms are in van der waals contact – R groups extend outwards from helix.

Pro will kink a helix (helix-breaker)
Not Gly—too many other conf (favor b sheet)
Steric clashes between large/branched side chains disfavor helix formation (I, Y)

With all C=O pointing towards C-term end, helix develops overall charge. N-term is +. C-term is -.

http://www.youtube.com/watch?v=Y24qTh4uUHU

Front 35

Secondary Structure

Back 35

Beta Sheets

In β Sheet, the H-bonds occur between neighboring peptide chains.
Antiparallel β Sheet, the two chain run in opposite direction
Parallel β Sheet, the two chain run in the same direction

In β Sheet, the side chains of two successive side chains locate on the opposite side of the sheet.


β Sheet in proteins can contain between 2 – 22 polypeptide strands with average of 6 strands.
Each strand can have up to 15 residues, average 6 residues.
Parallel β Sheet rarely has less than 5 strands, which suggests that antiparallel β Sheet is more stable than parallel β Sheet (distorted H bonds)
7.0 A between R groups (pleat)

Front 36

Tertiary Structure

Back 36

3o structure is a collection of 2o structures in motifs or domains


3o str dependent on side chain interactions:
Disulfides (covalent) between cysteines
(also cross-links between metal ions and ligands Cys, His)
Salt bridges (ionic) between acidic and basic residues
H-bonds between polar residues
Hydrophobic interactions between nonpolar residues
Sum of lots of noncovalent interactions hold proteins together in 3D fold.

Front 37

Globular Proteins

Back 37

A hydrophobic core and hydrophilic surface

Hydrophobic effect drives protein folding

Front 38

Quaternary structure

Back 38

Multiple subunits held together by primarily non-covalent interactions

Same as tertiary structure interactions

Front 39

Peptide bond hydrolysis

Back 39

Condensation reaction (elimination of water)
Very slow reaction – thermodynamically favorable, but kinetically controlled
Peptide bond has half-life of 20 years

Change rate of reaction: increase temperature, increase substrate concentration, add a catalyst
Enzyme = protein-based catalyst

Front 40

Enzymes

Back 40

General properties of enzymes:
A. Enzyme catalyzed reactions are 10^6 – 10^12 times faster than the uncatalyzed reactions and at least several orders of magnitude higher than chemically catalyzed reactions.
B. Mild reaction condition: enzymatic reactions occur at < 100 C, atmospheric pressure, and near neutral pH. Chemical reactions normally need more stringent conditions: high temperature, high pressure and extremes of pH.
C. Reaction specificity: for both substrate and product -- distinguish small structure perturbations on substrates. rarely have side products (even stereoisomers)
D. Regulation: enzymatic reactions are normally regulated. Common methods used to regulate enzymatic activity: allosteric regulation, post-translational modification of proteins, and up or down regulation of enzyme concentrations.

Ex. Carbonic anhydrase CO2 + H2O -->H2CO3

Front 41

Enzyme-Substrate Specificity

Back 41

Forces used in enzyme/substrate interaction: similar to the forces used in protein folding
A. van der waals interactions
B. H-bonding
C. electrostatic interaction
D. hydrophobic interactions

Factors contributing to the specificity in substrate binding:
A. geometric complementarity (the shape of the protein binding pocket matches with the shape of the substrate)
B. electronic complementarity (negatively charged area on the substrate face the positively charged area in the enzyme)
(lock and key)
C. induced fit: binding of the substrate can induce conformational changes of the enzyme to accommodate both geometric and electronic complementarity.

Geometric specificity more stringent than stereospecificity

Enzymes do have some degree of flexibility. For examples, ethanol dehydrogenase can also take methanol or isopropanol as the substrate at a much lower turnover rate.
Chymotrypsin can take both peptide and esters as the substrate—however does have geometric specificity aromatic (Y, W, F) just upstream from cleavage site (R-group)

Many enzymes permissive—but how permissive is question: Lock-and-key vs. Induced fit “spectrum”

Front 42

Classifications of Enzymes

Back 42

There are six major classes of enzymes:
oxidoreductase: redox reaction
transferase: transferase reaction
hydrolase: hydrolysis
isomerase: isomerization
lyase: group elimination
ligase: bond formation at the expense of ATP hydrolysis

Front 43

Cofactors

Back 43

Cofactors can be either metal ions or small organic molecules.
Common metal ions in enzymes: Ca2+, Mg2+, Fe3+, Zn2+, Cu2+. The toxic effect of some metals (Cd2+, Hg2+) are due to the replacement of other essential metals by them.

Cosubstrate = transient association
Prosthetic grp = permanent association

Apoenzyme: enzymes after removing the cofactors; holoenzyme: enzyme-cofactor complex.

Front 44

Coenzymes and Vitamins

Back 44

The compounds present in organism’s diet, which are used as the precursors for coenzyme biosynthesis, are called vitamins

Example: Nicotinamide or nicotinic acid are the precursors for NAD+. Supplementation of human diet with nicotinamide relieves the human dietary disease, pellagra (diarrhea, dermatitis, and dementia)

All vitamins used to coenzyme synthesis are water soluble vitamins. The water insoluble vitamins are not used in coenzyme biosynthesis.

Front 45

Transition State

Back 45

ΔG‡ and ΔG represent two different concepts in chemistry.

ΔG‡ the free energy of activation. It is the factor governing the reaction rates (Kinetics).

ΔG is the free energy of the reaction. It is the factor governing the feasibility of the reaction (Thermodynamics).
ΔG < 0, reaction will go to the forward direction
ΔG = 0, reaction is at the equilibrium
ΔG > 0, reaction will go to the reverse direction

Front 46

Rate enhancement: ΔΔG‡

Back 46

Transition state theory: for chemical reactions, there exists a state in which a bond is in the process of breaking and another bond is in the process of forming.
ΔΔG≠ decrease by 1.36 kcal/mol (5.71 kJ/mol) will increase rate by 10 fold
10 = e^(5.71/8.3145 J/K mol * 298 K)
V = e^(ΔΔG≠ /RT)

One important H-bond can increase rate by > 100 fold. (Hbond ~20)
10^6 fold increase with ~34 kJ/mol

Small decrease of ΔG≠ can result in dramatic rate acceleration
Accelerates both fwd and reverse reactions – allows rxn to reach equilibrium faster

Front 47

Mechanisms of enzyme catalysis

Back 47

Preferential binding of transition state
Proximity effects

Acid-base catalysis
Covalent catalysis
Metal-ion catalysis

Front 48

Transition State Theory

Back 48

Transition state theory predicts that enzyme has higher affinity to transition state relative to the ground state or the product.

If a compound mimics the transition state, it should have very high affinity to the enzyme and can be great inhibitors.

Based on the transition state theory, the enzyme has preferrential binding to the transition state, which leads to the dramatic decrease of the activation energy.

Front 49

Preferrential Binding of Transition State

Back 49

The most important contribution to the rate acceleration in enzyme catalysis is the preferential binding of enzyme to the transition state.

Stabilizing the transition is the most significant one. Every 1.36 kcal/mol decrease in activation free energy increases the rate by 10 x. Millions of fold increase can be easily achieved.

Front 50

Proximity and Orientation Effects

Back 50

Same as effects seen with linking reactants in organic chemistry

Enzyme can enhance rate by confine the substrate:
Bring together the substrate or put the substrate to enzymatic catalytic groups (5 x increase in rate)
Proper orientation (e.g. SN2 reaction needs the linear alignment of the reaction groups; slight deviation from that can significant decrease the reaction rate) (5 x increase in rate)
Charge groups can stabilize the transition state (electrostatic catalysis); they can also be used for proper orientation.
Freeze the translational and rotational motions (107 fold) (Entropic cost—made up with binding energy of ES)
In the example, by simply coupling the substrate and the catalyst (imidazole), rate can be increased by 24 fold.

Front 51

Factors that Influence Rate Acceleration in Catalysis

Back 51

In summary, the following factors contribute to rate acceleration in catalysis:
A. proximity (local concentration change after binding)
B. orientation of the substrate
C. strains generated in binding
D. desolvation after binding to the enzyme active site
E. Most importantly, the preferential binding of the enzyme to the enzyme active site.

Front 52

Acid-Base Catalysis

Back 52

General acid catalysis: the process in which proton transfer from an acid lowers the free energy of a reaction’s transition state.

General base catalysis: the process in which the abstraction of a proton by a base lowers the free energy of a reaction’s transition state.

Concerted acid/base catalysis: the process in which both general acid and general base are involved

Front 53

pH Dependence of Enzymatic Reactions

Back 53

This pH dependence curve indicates that the reaction needs both general acid and general base

The left hand side is a general base (dec pH protonate = block needed abstractor)
and the right hand side is a general acid (inc pH deprotonate = lose needed H)
Ex: Inflection point (pKa) ~ 5.8 and ~ 7.6
Deprot His = gen base
Prot His = gen acid
pK 4 might be Asp or Glu; pK of 6 might be His; pK of 10 might be Lys.
**microenvironments

The pH titration curve can also be just half of the above model.

Front 54

Covalent catalysis

Back 54

Covalent catalysis: it accelerates the reaction rates through the transient formation of a catalyst-substrate covalent bond (covalent substrate)
(nucleophilic catalysis when nuc on catalyst rxts with electrophilic on substrate)

In acetoacetate decarboxylation reaction, the amine of a lysine residue function as the base to form Schiff base (Imine)

Front 55

Covalent Catalysis

Back 55

The nucleophilicity is related to the basicity of the functional group (note nucleophiles are deprotonated).
For biological nucleophilic groups, they are either negatively charged or neutral molecules with lone-pair electrons.

Good covalent catalysis must balance between nucleophilicity and the ability as a good leaving group, which means that they need to have high polarizability (such as imidazole in His and thiol in Cys)
**more stable the bond formed, the harder it is to decompose.
Nucleophilicity for starting reaction; good lvg group to release product

In addition, coenzymes (e.g., thiamine pyrophosphate and pyridoxal phosphate) function as covalent catalysts.

Intermediate (covalent adduct) = different (and lower) path through reaction (ie now two smaller ΔG‡)
**Differentiate between intermediate and transition state!

Front 56

Metal-ion catalysis

Back 56

1/3 of the enzymes are metallo-enzymes. Common metals in enzyme include: Na+, K+, Mg2+, Ca2+, Zn2+, Fe2+/3+, Co2+, Ni2+, Cu2+, Mn2+

Structural roles: Na+, K+, Mg2+, Zn2+,

Catalytic roles: Mg2+, Ca2+, Zn2+, Fe2+/3+, Co2+, Ni2+, Cu2+, Mn2+

Roles of metal ions in catalysis: a) orientate the substrate in the enzyme active site; b) mediate oxidation-reduction reactions; c) electrostatically stablizing or shielding negative charges.

Example of Zn2+ in H2O activation (metals are normally more potent than proton in neutralizing negative charges. metal ligation to H2O can also significantly increase the H2O acidity)

http://www.youtube.com/watch?v=SsI-sQ6Ows8
H2O in carbonic acid anhydrase is activated by Zn2+, in which His64 facilitates this process. This is a very important process in buffer the human body

Front 57

Serine Proteases

Back 57

Serine proteases cleave peptide bonds (hydrolysis)
Catalytic triad: Asp, His, Ser

http://www.youtube.com/watch?v=ObivsZ93MNs

Front 58

Ex. Serine Proteases
Stabilization of transition state

Back 58

After the formation of the tetrahedron intermediate, peptide move deeper into the active site.

At the same time, there is a new set of H-bonds: the H-bond between oxyanion and the NH of Gly193 and Ser195 backbone.

The preferential binding of the transition state contribute significantly to catalysis.

Rate enhance: mutants of the triad 5 x 104; native enzyme 1010.

Front 59

Substrate specificity of serine proteases

Back 59

Chymotrpysin has a big pocket for aromatic residues and Ser189 at the bottom

Trypsin has Asp189 at the bottom to form charge-charge interactions.

Elastase: has filled pocket. Thus it take substrates with small side chains.

The substrate specificity is not merely controlled by one or two residue. For example, the change of residue Asp189 or Ser189 does not change chymotrypsin to trypsin. Instead, the residues used to form the pocket control the selectivity. (surface loops also replaced to convert trypsin to chymotrypsin-like)

Front 60

Inhibitors of Specific Serine Proteases

Back 60

By slight modification of the inhibitor, they can be tailed to target specific classes of Ser proteases.
Bulky aromatics for chymotrypsin
+ chg for trypsin

Trypsin, chymotrypsin, elatase = divergent evolution

Front 61

Reaction Kinetics

Back 61

Unimolecular (first order) = dependent on the concentration of only one substrate

Bimolecular (second order) = dependent on two substrate concentrations.

Front 62

Terms

Back 62

The model of enzymatic reaction can be roughly divided into two sections: binding and catalysis

A few important terms, reaction rate constants (forward and reverse reactions), equilibrium constants and dissociation constants.

Front 63

Rate Dependence

Back 63

At high substrate concentration, [ES] = [E]0.

ν0 = k2 [ES] = k2 [E]0.

Front 64

General Features of Enzyme Kinetics

Back 64

At low substrate concentration, it is pseudo 2nd order reaction ν is propotional to both [E]0 and [S].

At high substrate concentration, ν is propotional to [E]0, but independent of [S].

Initial rate and [S] relationship, hyperbolic curve.

Front 65

Back 65

Definition of the symbols used in enzymatic reactions: S (substrate), P (product), ES (enzyme/substrate complex, Michaelis complex)

The product formation rate depends on [ES] and k2.

Without any assumption, this will be a very difficult equation.

Under the following initial rate so that the reverse reaction can be ignored

A. the rate limiting step is after the substrate binding (k-1 >> k2).
B. Rapid equilibrim asumconditions, Michaelis-Menton is derived.
C. [S] >> [E], thus, the amount of S present in the form of ES can be ignored.
D. Steady state assumption: the concentration of ES remains constant for a long time, which means the formation and decomposition rate are the same (d[ES]/dt = 0).

Front 66

Michaelis-Menton analysis

Back 66

Assumptions:
k2 is rate-limiting:
k2 >>> k-2 and
k-1 >>> k2


d[ES]/dt = 0 (Steady state)
[E]T = [E] + [ES]

Front 67

Michaelis-Menton Variables

Back 67

Vmax: the maximal velocity of a reaction. It occurs a high substrate concentration and all enzymes are saturated by the substrate.

Km: Michaelis constant and it has the concentration unit. The concentration of the substrate that gives Vmax/2.

Km = Kd + k2/k1. Km is very close to Kd when k2 << k-1.

The most tricky one is Km. Many people get confused with Kd.

In many cases, Km is very close to Kd and used to determine the tightness of the interaction between enzyme and substrate

http://www.youtube.com/watch?v=W9o4cQvbShU

Front 68

Low-substrate concentration ([S] << Km )

Back 68

v = k2 [ES]

At low substrate concentrations, the reaction rate has a linear dependence on substrate concentration.

Front 69

High-substrate concentration ([S] >> Km )

Back 69

v = k2 [ES]


At high substrate concentration, the enzymatic rate is substrate concentration independent.

Front 70

variables

Back 70

Km: Michaelis constant and it has the concentration unit. The concentration of the substrate that gives Vmax/2.
Km = Kd + k2/k1. Km is very close to Kd when k2 << k-1.

kcat = Vmax/[E]T. Turnover number: this number can change by orders of magnitude. In the above case, kcat = k2. kcat is enzyme concentration independent while Vmax is concentration dependent.