Bioc 440 Exam 2

Protein structures are not

Static

Ligands are things

That reversibly binds to a protein

Apo

No ligand

Substrate bound

With ligand

Kd =

[P][L] / [P * L] (units of concentration M)

Theta (Y)

Fraction of occupied binding sites [occupied] / [total]

The lower the Kd

The lower the concentration of ligand required to bind to the protein (higher affinity)

Theta equation

[P*L] / ([P] +[P*L]) = [L] / ([L] +Kd)

The concentration at which half the sites are occupied [L] is

Kd, the dissociate equilibrium dissociation constant

Smaller Kd means

Tighter binding, higher affinity → generally need less ligand to fully occupy binding sites

Larger Kd means

For a given L generally close to cellular [L] (Small changes in ligand concentration gives large changes in occupied sites)

Myoglobin

Alpha helical + 1 heme group

Hemoglobin

O2 storage → 2 histidines: 1 binds Fe in core, one binds oxygen. These 2 positions are INVARIANT

Mb has HIGH

O2 affinity (storage protein)

Moving to the left on Mb curve means

Myoglobin is giving up occupancy of oxygen

Hb’s function

Delivery of O2 from lungs to tissue

Why is hemoglobin good at binding when it has low affinity

Cooperativity of the 4 heme groups

Cooperativity

Communication bt subunits influences affinity basis for more switch-like behavior

Hb binding curve is

Sigmoidal

Hb optimized for binding in

Lungs and releasing in tissues

p50

Measure point on curve where sites are half filled and what is the cooresponding oxygen concentration

Hemoglobin exists in two states called

T and R

T state affinity

Terrible affinity

R state affinity

Really strong binding

[T] / [R] ratio

Function of pO2

Theta (O2) measured under a given condition is a function of

The ratio of states pO2 up; [R] / [T] up; eff p50 down

Positive cooperativity

Making binding at the next site more favorable

Expression for simutaneous multiple binding events

P + NL → ← P * Ln

Binding one or more ligands

The steepest the sigmoidal curve can be for the number of binding sites in a protein

A hill coefficient of exactly the number of binding sites means

A protein binds all of the ligand at the same time (not possible: theorhetical limit)

Positive cooperativity =

Higher the slope

T state

Deoxy, weakly bound to oxygen, “tensed”

R state

Oxy, tightly bound to oxygen, “relaxed”

[T] / [R] determines

p50 effective

O2 homotropic for

O2 binding

p50

If n=1, p50=pO2 fill half the sites. If n>1, p50 > pO2 fill half the sites

Allosteric protein

One with another conformation induced by binding or post-translational modification

Ligands that affect binding at another site =

“Effectors” or “modulators”

Effector same as ligand whose binding is affected

Homotropic

Effector different than ligand whose binding is affected

Heterotropic

Structural effects of O2 binding relative to heme

1. F-helix adjusts relative to heme (close packing, effects propogate)

2. T-state stabilized by ionic interations at interface bt subunits

3. Adjustments in F-helix on O2 binding alter subunit-subunit interactions and stabilize R-state

H+ increase causes

[T] / [R] to increase, affinity down

pH decrease via CO2 production

Lower pH in tissues promotes dissolution

Bohr effect

Carbon dioxide and hydrogen ions lower hemoglobin’s affinity to oxygen

How does protonation favor the T state?

His+ stabilized by nearby negative charge; in T state HIs has unusally high pKr

Lower affinity means you’re spending more time in

T state

CO2 binds at the ___ in hemoglobin

N terminus

CO2 releases an H+

Additional Bohr effect

CO2 binding promotes the T state

new salt bridges → stabilize T state

Respiring tissue: CO2 up

O2 release

Lungs O2 is high: CO2 low

O2 binding

BPG binds to hemoglobin and promotes /

stabilizes the T-state

Circulating BPG levels increase with

High altitude (change in the oxygen concentration in the atmosphere)

BPG has one

Binding site per hemoglobin

BPG shifts the curve to the

Right

BPG is negatively charged and binds

To positively charged site in the T-state → site disappears in the R-state

CO poisoning

CO binds to the heme group in the same position as O2 with higher affinity than O2

CO Hb has slow

Dissociation → lowers number of O2 binding sites

Hb * CO binds O2 with a ____ affinity than Hb

Higher

Delta G double dagger

Difference between ground and transition state. Rate of rxn depends on it

Enzymes do not change

The overall dG of the reaction

Rate enhancement equation

Rate (cat) / rate (uncat)

Substrate

Molecule upon which enzyme reacts

Active site

Where the enzyme chemistry occurs

Cofactor/coenzyme

Groups that bind to enzyme that can be important for its catalysis

Holoenzyme

Enzyme that has cofactors/coenzymes bound to be able to carry out its function

Enzyme naming general rule

“Something” + “ase”

Velocity

Number of substrate molecules converted per unit time

DG double dagger in the exponent

Small differences give big changes in rate constant (kcat)

Substrate orientation (proximity) and entropy reduction

Increases probability of reaction to occur

Enzymes can enhance rxn rate without wasting ATP by

Spurring catalysis when all substrates present even when only bound to ATP

Acid base catalysis

Acidic/basic groups in active site accept/donate H+ to stabilize an unstable species

pKr depends on the relative stability of

HA and A- and local environment is important

Covalent catalysis

Reaction includes and intermediate

Metal ion catalysis

Metal ions in active sites bind and orient substrate, stabilize charged intermediate, perform redox chem

Most enzymes use

>1 strategy

We study enzymes and substrates in

Purified form

Measure reaction rate by enzyme assay

Need singal proportional to [S] or [P] concentrations

The rate of production of product is proportional to the concentration of

Substrate → high concentration of substrate pushes towards products faster

Plateau

Reaching equilibrium

Km

Half the Vmax

Michaelis Menton Equation describes

Reaction rate (v0) as a function of [S]

Enzyme catalyzed reaction

E + S → E * S → E + P

E+S → E*S

Fast

E*S → E + P

Slow

Enzyme concentration is limiting compared to substrates, so

[S] is effectively constant early in the rxn

In early reaction [P] is

0

k2 is

Slow relative to k1 and k-1

E*S is in

Steady state (NOT EQUILIBRIUM); means E*S does not change w/time

Total enzyme concentration

[E]T = [E] + [E*S]

[E*S] Equation

[E*S] = [E]T[S] / (Km + [S])

Maximum rate possible if

[E]T = [E*S]

Vmax =

k2[E*S]

Define k2 =

kcat

Vmax when all

E in the E*S complex

At low [S]

[S] « Km

At high [S]

[S] > Km

kcat is the “turnover number”

max number of rxns/active site/unit time

Catalytic efficiency

kcat/km

Competitive inhibition

Molecule that looks like enzyme instead binds and inhibits