Biochemistry Lecture Notes Chapter 8

Front 1

What are enzymes?

Back 1

Accelerate biochemical reactions by facilitating the formation of the transition state
-Michaelis-Menten model accounts for the properties of many enzymes

Front 2

What is free energy?

Back 2

-useful thermodynamic function that accounts for spontaneity but NOT RATE OF REACTION

∆G products - ∆G reactants
independent of path

Front 3

When is a reaction spontaneous?

Back 3

∆G<0, rxn is spontaneous
∆G=0, system is at equilibrium
∆G>0, nonspontaneous

Front 4

∆G equation

Back 4

∆G = ∆Go + RT ln ([C][D]/[A][B])

Front 5

What is ∆Go? Give equations.

Back 5

∆G=0, so lose it from equation.
∆Go = -RT ln([C][D]/[A][B])
Keq = ln([C][D]/[A][B])
∆Go = -RT ln Keq = -2.303RT log base 10 (Keq)
substituting R and T....
Keq = 10^(-∆Go/1.36)

Front 6

What are the values for R?

Back 6

1.987x10^-3 Kcal/molK

Front 7

Can a reaction be made spontaneous?

Back 7

Yes. If deltaGo is positive (nonspontaneous), then the reaction can be made spontaneous by ADJUSTING THE CONCENTRATION OF REACTANTS AND PRODUCTS -- this is why we couple reactions to form metabolic pathways.

Front 8

How does Gibbs Free Energy relate to transition states?

Back 8

The energy of activation, ∆Gdagger, is the difference in free energy between the transition state and substrate

Front 9

How do enzymes accelerate reactions?

Back 9

-They accelerate the attainment of equilibria, but do not shift their positions.
-activation energy/barrier is lowered by enzymes

Front 10

What are characteristics of active sites of enzymes?

Back 10

-is a 3D cleft that binds the substrate and cofactor
-relatively small part of the enzyme's total volume
-subtrates bind by multiple weak attractions
-specificity of binding depends on the precisely defined arrangement of atoms in active site

Front 11

What are the E-S binding models?

Back 11

Lock and key: active site of unbound E is complementary to S

Induced fit: E changes shape upon S binding

Front 12

How does reaction rate depend on ∆Gdagger?

Back 12

V = v[Sdagger] = (kT/h)[S]e^(-∆Gdagger/RT)

Front 13

Where is the evidence of the formation of an E-S complex?

Back 13

1. x-ray crystallography: high resolution images shows it!
2. ES complex formation causes spectroscopic changes--> can see with tryptophan synthetase
3. reaction rate increases with increasing substrate concentration
4. max velocity attained -- suggested formation of ES complexes

Front 14

What factors influence product formation?

Back 14

time and substrate concentration

Vo almost linear to [S] when [S] is small
Vo nearly independent of [S] when [S] is large

Front 15

What is Km?

Back 15

Km = (k-1 + k2)/k1 = [E][S]/[ES]

Front 16

What does Km tell you?

Back 16

it measures the affinity of substrate for an enzyme.
high Km: low affinity
low Km: high affinity and tighter binding!

independent of substrate and enzyme concentration

Front 17

Michaelis-Menten Equation

Back 17

Vo = Vmax [S]/([S] + Km)

Front 18

What is Vo?

Back 18

Vo = k2[ES]

Front 19

What is Vmax?

Back 19

Vmax = k2[Et]

Front 20

how is reaction velocity related to [S] and Km?

Back 20

[S]<Km, Vo = Vmax ([S]/Km), rate is directly proportional to the substrate concentration

[S]>Km, Vo = Vmax, rate is independent of [S]

[S] = Km, Vo = Vmax/2

Front 21

Double reciprocal plots for enzyme kinetics

Back 21

Y axis: 1/Vo, X axis: 1/S
Y intercept = 1/Vmax
X intercept = -1/Km
Slope = Km/Vmax

Front 22

What is Kcat?

Back 22

the turn over number (T.O.N.) of an enzyme, or the number of substrate molecules converted into product by an enzyme in a unit time when the enzyme is fully saturated with substrate

equal to k2

Front 23

fES

Back 23

fraction of active sites filled

fES = V/Vmax = [S]/([S] + Km)

Front 24

how do you measure catalytic efficiency?

Back 24

in the body, substrate is rarely ever in excess, so omit the S from the denominator, and V=Vmax=k2Et

So, Vo = (k2/Km)[Et][S], where k2/Km is a measure of catalytic efficiency because it accounts for the rate of catalysis and the strength of E-S interaction

can use this to compare an enzyme's preference for substrates

Front 25

What is Kcat/Km

Back 25

Rate constant for th interaction of S and E
measure of catalytic efficiency
cannot be higher than 10^9 (sM)^-1, the diffusion controlled encounter of E and S
--> at upper limits, kinetic perfection

Front 26

What are the biochemical reactions with multiple substrates called?

Back 26

1) sequential displacement: ordered and random

2) double displacement (ping pong)

Front 27

What is sequential displacement?

Back 27

*A and B bind to E before P and Q are released

1) ordered: A and B bind to E in a defined sequence
example: lactate dehydrogenase. NADH/NAD+ binds then pyruvate/lactate. COFACTOR BINDS FIRST!

2) random: A and B bind to E indiscriminately
example: creatine kinase. catalyzes creatine to phosphocreatine, an important energy source in muscle

Front 28

Whta is double displacement?

Back 28

Ping Pong!

*P and Q may be released from E before A and/or B

defining feature: substituted enzyme intermediate

example: aspartate aminotransferase, catalyzes transfer of amino acid from aspartate to alpha-ketoglutarate

Front 29

How are enzymes inhibited?

Back 29

1) Reversible Inhibitors
A)Competitive
B) Noncompetitive

2) Irreversible

Front 30

What is a competitive inhibitor?

Back 30

-Reversible. Resembles substrate, binds to active site

-Only binds free E

-Increasing S overcomes inhibition

-Vmax unchanged
-Km increased

Front 31

What is a noncompetitive inhibitor?

Back 31

-Reversible. Binds to site different from active site

-Can bind to E or ES complex.

-Increasing S can not overcome inhibition

-Vmax decreased
-Km unchanged

Front 32

What are the types of irreversible inhibitors?

Back 32

1. Group specific reagents: modify functional groups within catalytic site

2. Affinity labels/reactive substrate analogs: molecules that are structurally similar to the substrate, covelaently bind active site residues. More specific for the active site than group specific reagents.

3. Suicide inhibitors: generates a chemically reactive intermediate that inactivates E through covalent modification. Most specific of the 3!

Front 33

What are examples of group specific reagents?

Back 33

1. DIPF: reacts with Ser residues

2. Iodoacetamide: alkylates a cysteine residue

Front 34

What are examples of affinity labels?

Back 34

1. TPCK: binds to active site and irreversibly reacts with histidine, inactivates chymotrypsin

2. 3-bromoacetol: binds to TPI and modifies E irreversibly.

Front 35

What are examples of suicide inhibitors?

Back 35

Monoamine oxidase is an enzyme that deaminates neurotransmitters such as dopamine and serotonin

Suicide inhibitors:
1. N,N,-dimethylpropargylamine: monoamine oxidase is inhibited. covalent modifications to the flavin prosthetic group after the inhibitor has been oxidized.

2. (-)deprenyl is a suicide inhibitor of monoamine oxidase, used to treat Parkinson disease and depression