pH and fumarase

pH and fumarase

Forward reaction: B2 has to accept a proton from water What if pH is too low? What if pH is too high?

This week’s lab notes

• You want to know the total activity of each fraction

slope (abs/min) → rate (mol/min)

Think of this rate as # units of fumarase activity in the volume you assayed (eg. you may have added 10 L to 990 L assay buffer).

But, you have to correct for the total volume of the sample. (eg. you may have applied 10.4 mL of crude to the column)

From abs/time

SampleTotal

Volume(mL)

Rate(mol/min)

VolumeAssayed

(mL)

TotalActivity

(mol/min)

Yield(%)

Crude 10.4 0.25 0.010 260 100

FT 14.1 0.05 0.010 70.5 27.1

Pooled Elutions

3.2 0.31 0.010 99.2 38.2

How much of thatsample you testedfor activity (~10L)

assayedvol

voltotalRate

.

.

Sample’s total activity vs. crude’s

Plan:

• Exam over Ch. 4, 5.1 plus Expt 3 weeks 1 and 2 (fumarase purification and ion exchange)

• Today: finish up 5.1 (Hb), start Ch. 6

Hemoglobin

• Cooperative binding– Binding of O2 at one subunit affects the

oxygen affinity of other subunits

• Allostery: – Regulation by reversible binding at a site

other than the active site– “Allosteric activation”– O2: homotropic allosteric activator

Another allosteric modulator

bisphosphoglycerate (BPG)

• Heterotropic allosteric inhibitor

• Binding of Hb•BPG has a lower affinity for O2 than does Hb

• Enhances release of O2 in the tissues

One BPG molecule per tetramer

Pushes T ↔ R equilibrium tothe left

T state

R state

High affinity for BPGStabilized by BPG

Low affinity for O2

High affinity for O2

Stabilized by O2

Low affinity for BPG

Enzymes

• Biological catalysts– High specificity and efficiency relative to

inorganic catalysts, for example– Participate in reactions, but no net change– Lower the activation energy– Do not change equilibrium (get there faster)

Enzymes

• Almost exclusively proteins (some RNA, others?)

• Protein may require cofactor(s)

(non-amino acid functional groups)– Apoenzyme: protein alone– Holoenzyme: protein + functional group

– Metals, nucleotide-containing cofactors, etc.

Enzymes

• Usually noted by “-ase” at the end– DNA polymerase, protein kinase, etc.

• Many enzymes have a common ‘trivial’ name– Fumarase, hexokinase, lysozyme, etc.

• All enzymes have a systematic name– Substrate(s) and reaction catalyzed

• Fumarase = “fumarate hydratase”• Hexokinase = “ATP:glucose phosphotransferase”

Enzymes

• Some common classes of enzymes– Kinases transfer phosphate (usually from

ATP) to another substrate– Phosphatases remove (hydrolyze) a

phosphate– Polymerases string together nucleotides– Proteases cleave peptide bonds– Oxidoreductases transfer electrons between

substrates

Drugs often modulate the action of enzymes

CYCLOOXYGENASE

aspirin

www.3dchem.com

Arachidonic acid

Prostaglandin H2

Enzymes speed up biological reactions

H2CO3 → CO2 + H2O

10,000,000x faster + carbonic anhydrase

EN

ER

GY

(G

°)

REACTION PROGRESS

G < 0

Reaction should bespontaneous

Equil should favorproducts

Biological reaction:sugar + oxygen ↔ CO2 + water

Reactants (R)

Activation energy

EA

Kinetic barrier to reaction

High energy “Transition state”Intermediate between R & P

Products (P)

The energy barrier is critical for life

• Potentially deleterious reactions are blocked by EA

– Complex molecule degrading to simpler constituents

http://asm.wku.eduhttp://encyclopedia.quickseek.com/

DNAnucleotide

How do enzymes speed up reactions?

• Lower the activation energy

• Decrease the energy barrier

2H2O2 → 2H2O + O2

Isolated: EA ~ 86 kJ/molIn the presence of catalase: EA ~ 1kJ/mol

Hydrogen peroxide

Binding of substrate to enzyme creates a new reaction pathway

http://w3.dwm.ks.edu.tw/

An enzyme changes EA NOT G

Affects RATE, not EQUILIBRIUM

Without enzyme

With enzyme

EA = G‡

How is EA lowered?

• Enzyme’s ‘goal’ is to reduce G‡

• Two ways enzymes can affect G

– Improve H– Improve S

EA =G‡ = H - TS

G‡ = Gtrans.state – Greactants

Enzymes alter the free energy of the

transition state

enthalpy entropy

-

Example: More favorable H

A B

AOHBH

A BH+

+ H2O

+OH-

+

Charge unfavorableUnstable transition st.

A BH+

Ionic interaction stabilizesthe positive charge

OH-

Example: More favorable S

Two moleculesMore ‘freedom’Higher disorder (high S)

One moleculeLower disorder (low S)Unfavorable entropically

ENZYME

Example: More favorable S

Enzyme/Reactant COMPLEX

Essentially a single molecule

ENZYME

Enzyme/Transition state complex

Still a single molecule

Not much difference entropically

Remember

1. Enzymes lower the energy barrier

2. Decrease EA (G‡)

3. Provide an environment where:

• Transition state is stabilized (lower enthalpy)• Change of disorder (entropy) is minimized