1. H20 interacts with and dissolved hydrophilic biomolecules
2. H20 dissociates ionic compounds (ie: NaCl)
3. H20 causes nonpolar & nonionic (hydrophobic) and amphipathic biomolecules to cluster
4. Participates in biochemical reactions
5. Major componant of urine
6. Major componant of blood
7. Regulates temperature homeostasis

- a variable side chain (-R)

- a carboxyl group (-COOH)

- an amino group (-NH2)

- a hydrogen atom (-H)

All bound to a central carbon atom

• composed of hydrocarbons
• No double bonds
• Found on proteins that are aqueus in solutions

• can have a double bond
• no +Ve or -Ve charge

• Can be acidic or basic

- Acidic sidechains have an -OH somewhere

- Basic sidechains have can accept an electron

In the equation:

pK = -log₁₀K

... What is pK??

HA ↔ H⁺ + A

The two equations that indicate the strength of a bond between H⁺ and A^- are:

1.  Dissociation constant

K_d= ([H⁺][A^-]) / [HA]

2.  Affinity constant

K_aff= [HA] / ([H⁺][A^-])

In the Henderson-hasselbach equation ...

what is [A^-]/[HA]?

1. Ion exchange chromotography
2. Calorimetric or flurometric visualisation/quantification of amino acids

Explain calorimetric/flurometric visualisation/quantification?

How is it used for diagnosis??

• Dyes react differently and bind to different amino acid functional groups. This binding causes a coloured or flurescent derivative being formed.

• Specific AA's can be identified according to the dye colour

• Clinical disorders associated with high concentrations of specific AA's can be diagnosed using this method

Ie: PKU = high levels of phenalanine

     Cystinuria = high levels of cysteine

• Single AA chain
• AA's bonded together via peptide bonds
• Can be hydrolysed (broken down) chemically with strong acids or enzymatically by proteases

• Folded structure
• H-bonds
• Two types of folds:
  • α-helix
    • twists clockwise
    • sidechains stick out
  • β-pleated
    • chains lay side-by-side
    • H-bond between carbonyl group of one chain and amino group of another chain
    • Antiparallel

• 3D conformation of a SINGLE polypeptide
• Interactions between groups that are far away from eachother
• Interactions include:
  • Disulfide bonds
  • H-bonds
  • Ionic interactions
  • Hydrophobic interactions

• Arrangement of subunits in a protein that contains more than 1 polypeptide

What is DENATURATION?

How can it be caused?

The unfolding of  a polypeptide due to the breakage of bonds (other than peptide bonds).

Caused by:

• heat
• detergents
• polar solvents
• extreme pH
• reducing agents
• inorganic salts
• heavy metals

1. Oxidoreductases
2. Tranferases
3. Hydrolases
4. Isomerases
5. Lyases
6. Ligases

• Catalyze oxidation-reduction reactions
• Subclasses:
  • Oxidases
  • Reductases
  • Dehydrogenases

• Catalyse transfer of groups between DIFFERENT molecules (intermolecular transfers)
• Subclasses:
  • Transaminases
    • transfer amino groups
  • Kinases
    • transfer phosphate groups

• Catalyze the cleavage of bonds by the addition of water
• Subclasses:
  • Proteases
  • Lipases
  • Nucleases

• Catalyse the breakage of C-C, C-S and some C-N bonds

• Catalyse the transfer of groups WITHIN a molecule (intramolecular transfers)

• Form bonds between Carbon and O, S and N atoms
• Coupled to hydrolysis of high energy phosphates

How do chemical and enzyme catalysts differ?

(NOTE: 5 things)

1. Enzymes are specific to their substrate
2. Enzymes are affected by their environment
3. Enzymes are faster
4. Enzyme activity can be regulated
5. Enzymes do NOT generate side reactions

How are chemical and enzyme catalysts similar?

(NOTE: 3 things)

1. They BOTH increase the velocity of the reaction
2. Neither are used up in the reaction
3. They dont alter equilibrium

The amount of enzyme that converts 1 micromole of substrate / min at a given temperature, pH and [substrate]

UNITS: enzyme unit U or katal

V_o = V_max( [S]/(K_m+[S]))

What do we assume when using the Michaelis-Menten equation?

NOTE:

S + E ↔ SE → E + P

Where:

S + E ↔ SE   is the binding step

SE → E + P  is the catalytic step

1. [S] >> [E]     (more substrate than enzyme -> enzyme is saturated)
2. The catalytic step is irreversable
3. The catalytic step is slower than the binding step
4. Reaction is at a steady state (formation rate = breakdown rate)

S + E ↔ SE → E + P

Where:

S + E ↔ SE   is the binding step

SE → E + P  is the catalytic step

Michaelis-Menten equation:

V_o = V_max ( [S] / (K_m + [S]) )

What is:

V_max    ,    V_o       &    K_m

V_max= the moment in time when enzymes are saturated ... MAX VELOCITY

V_o = initial rate of reaction

K_m = substrate concentration at 1/2 V_max (UNIT: M)

HIGH K_m = Low affinity

LOW K_m = High affinity

What are the physiological mechanisms of enzyme regulation?

(NOTE: 5 types)

1. Allosteric control
2. Genetic control
3. Feedback control
4. Posttranslational modification mediated control
5. Intracellular compartmentation medicated control

What is an allosteric enzyme?

AND

Describe Allosteric enzyme control...

• An allosteric enzyme is a multisubunit protein that has one or more active sites
• A regulator binds to an active site and causes a conformational change in the shape of the enzyme.
• This change alters the affinity of the other binding site for a substrate

With allosteric control ... what is the regulator?

Do allosteric enzymes follow the Michaelis-menten kinetics?

• A regulator  can be either an
  • activator
    • increases enzyme activity
    • +Ve cooperativity
  • inhibitor
    • decrease enzyme activity
    • -Ve cooperativity
• Allosteric enzymes do NOT follow Michaelis-Menten kinetics
• They show a SIGMOIDAL curve

• Where the end product of a series of enzyme catalysed reactions can influence the activity of one of the earlier enzymes (ie: inhibit or activate)

1. Energy production/consumption
2. Energy transduction (conversion of energy from one form to another)

Biological systems follow the laws & principles of thermodynamics

What are laws 1 and 2??

• 1st law of thermodynamics:
  • Energy in the universe is constant ... it cannot be created or destroyed

Total energy_universe = total energy_{reacting system} + total energy_surroundings

• 2nd law of thermodynamics
  • At a given temp and pressure ... reactions proceed in direction leading to an increase in the entropy of the universe

Disorder_universe = Disorder_{reacting system} + Disorder_surroundings

It cannot be experimentally determined ... but it CAN be calculated using variables that can be measured

ΔG = ΔH - TΔS

Where:

• G = energy available for work at a given temp (T)
• H = ENTHALPY ... heat content of a reacting system

Whether a reaction can occur spontaneously or not

What does it mean if ΔG < 0 ??

• Reaction WILL occur SPONTANEOUSLY
• Energy is released
• Reaction is energetically FAVOURABLE

• Reaction will NOT occur spontaneously
• Energy MUST be comsumed for the reaction to occur
• Reaction is energetically UNFAVOURABLE

Remember: ΔG depends on the NATURE and CONCENTRATION of the reactants

What equation can we use to determine ΔG ?

How can we influence the direction of a reaction??

(two ways)

1. Removing product
2. Coupling an energetically unfavourable reaction to an energetically favourable reaction

1. Preparatory stage
   1. Priming
   2. Splitting
2. Payoff stage

1. Preparatory
   1. priming ------> reactions 1, 2 and 3
   2. splitting -----> reactions 4 and 5
2. Payoff stage ------> reactions 6, 7, 8, 9, 10

GLYCOLYSIS

REACTION #1

Glucose ------------------> Glucose-6-Phosphate

ATP is produced!

Enzyme: HEXOKINASE

Type: phosphorylation reaction

1st regulatory step:

• G-6-P acts an an allosteric inhibitor of hexokinase
  • ↑ G-6-P = inhibition
  • ↓ G-6-P = activation

GLYCOLYSIS

REACTION #2

Glucose-6-phosphate <-----------> Fructose-6-phosphate

Enzyme: PHOSPHOGLYCOISOMERASE

Type: isomerisation reaction

GLYCOLYSIS

REACTION #3

Fructose-6-phosphate ------> Fructose-1,6-bisphosphate

ATP is USED UP

Enzyme: PHSOPHOFRUCTOKINASE

Type: phosphorylation reaction

• REGULATORY SITE #2
  • PFK inbibited by ↑ATP
  • PFK activated by ↑ADP

GLYCOLYSIS

REACTION #4

Fructose-1,6-bisphosphate <-----> dihydroxyacetonephosphate AND

glyceraldehyde-3-phosphate

Enzyme: FRUCTOSE BISPHOSPHATE ADOLASE

type: cleavage reaction

GLYCOLYSIS

REACTION #5

Dihydroxyacetonephosphate <----->

Glyceraldehyde-3-phosphate

Enzyme: TRIOSE PHOSPHATE ISOMERASE

Type: Isomerisation reaction

GLYCOLYSIS

REACTION #6

Glyceraldehyde-3-phosphate <-----> 1,3-bisphosphoglycerate

NADH + H⁺ released

Enzyme: G-3-P dehydrogenase

Type: Oxidation-reduction reaction

BPG = HIGH ENERGY COMPOUND

GLYCOLYSIS

REACTION #7

1,3-bisphosphoglycerate <-----> 3-phosphoglycerate

ATP RELEASED

Enzyme: phosphoglycerate kinase

Type: kinase reaction

SUBSTRATE LEVEL PHOSPHORYLATION #1

GLYCOLYSIS

REACTION #8

3-phosphoglycerate <--------> 2-phosphoglycerate

Enzyme: PHOSPHOGLYCERATE MUTASE

Type: isomerisation reaction

GLYCOLYSIS

REACTION #9

2-phosphoglycerate ---------> phosphoenol pyruvate

Water realeased

Enzyme: ENOLASE

Type: Hydrolysis

PEP = HIGH ENERGY COMPOUND

GLYCOLYSIS

REACTION #10

Phosphoenol pyruvate --------> Pyruvate

2 x ATP RELEASED

Enzyme: Pyruvate kinase

Type: Phosphorylation

• REGULATION SITE #3
  • PK inhibited by ↑ATP
  • PK activated by ↑ADP

SUBSTRATE LEVEL PHOSPHORYLATION # 2

What are the two ways NAD⁺ stores are replenished?

What tissues use each strategy?

STRATEGY #1

As alcohol dehydrogenase converts acetaldehyde to ethanol ... NADH and H⁺ go in to the reaction and NAD⁺ is released

This strategy is used in yeasts and in the intestinal cells of humans

STRATEGY #2

As lactate Dehydrogenase converts pyruvate to lactate ... NADH and H⁺ go in to the reaction and NAD⁺ is released

This strategy is used in RBC's, lens & cornea of the eye, kidney medulla, testes and intensly exercising muscle

It is an enzyme composed of 3 parts

(a multienzyme complex)

1. By product inhibition (↑Acetyl CoA will inhibit PDH)
2. By covalent modification

The pyruvate dehydrogenase reaction is the reaction that is performed between glycolysis and the kreb cycle.

What are the products of this reaction??

• Acetyl CoA
• NADH
• H⁺
• CO₂

• 1 x GTP
• 1 x FADH₂
• 3 x NADH
• 1 x CO₂
• 3 x H⁺
• 1 x CoA

CITRIC ACID CYCLE

Reaction # 1

Acetyl CoA + Oxaloacetate -----> Citrate

H₂O inputted

Enzyme: CITRATE SYNTHASE

Type: condensation reaction

• REGULATION SITE # 1
  • CS inhibited by ↑ATP & ↑NADH
  • CS activated by ↑ADP

CITRIC ACID CYCLE

Reaction # 2

Citrate -------------> isocitrate

Enzyme: ACONITASE

Type: Isomerisation reaction

CITRIC ACID CYCLE

Reaction # 3

Isocitrate <---------> α-ketoglutarate

INPUT: NAD⁺

OUTPUT: NADH, H⁺ and CO₂

Enzyme: ISOCITRATE DEHYDROGENASE

Type: 1st Oxidation-reduction reaction

• REULATION SITE #2

CITRIC ACID CYCLE

Reaction # 4

α-ketoglutarate ------------> Succinyl CoA

INPUT: NAD⁺ and CoASH

OUTPUT: NADH and H⁺ and CO₂

Enzyme: α-KETOGLUTARATE DEHYDROGENASE

type: 2nd oxidative-carboxylation reaction

• REGULATION SITE #3

CITRIC ACID CYCLE

Reaction # 5

Succinyl CoA <--------> Succinate

INPUT: GDP and P_i

OUTPUT: GTP (first!)

Enzyme: Succinyl CoA synthetase

FIRST SUBSTRATE LEVEL PHOSPHORYLATION reaction

CITRIC ACID CYCLE

Reaction # 6

Succinate <---------> Fumarate

INPUT: FAD

OUTPUT: FADH₂

Enzyme: SUCCINATE DEHYDROGENASE

OXIDATION REACTION #3

CITRIC ACID CYCLE

Reaction # 7

Fumarate <--------> Malate

INPUT: H₂O

Enzyme: Fumarase

Type: hydration reaction

CITRIC ACID CYCLE

Reaction # 8

Malate <-----------> Oxaloacetate

INPUT: NAD⁺

OUTPUT: NADH and H⁺

OXIDATION REACTION #4

1. Transfer of a high energy phosphate group from a substrate to ADP -------> to produce ATP

2. By useing the energy released from breaking a high energy substrate to drive the synthesis of a phosphoanhydride bond

3. By using the energy generated by the transfer of electrons from the reduced form of coenzymes of substrate dehydrogenases to oxygen