Enzymes

Question 1

Structure of Enzymes

Answer 1

active site = small amount of amino acids with catalytic side chains that come together in tertiary structure and form a cleft. binds the substrate noncovalently bind the substrate STRONGLY

*nonpolar and excludes water

other amino acids form a scaffold to keep active site in the correct conformation

Question 2

Induced fit model

Answer 2

enzyme is inactive until the substrate binds to it and induces a conformational change which activates the enzyme

Question 3

lock and key model

Answer 3

enzymes have a rigid, predefined active site that perfectly matches up with the substrate and forms the ES compplex

this is wrong! doesn’t explain why some substrates perfectly fit into enzymes and don’t react

Question 4

How do enzymes accelerate the rate of reactions?

Answer 4

Enzymes bind to the substrate during the transition state and create a new reaction pathway with a lower activation energy for the reaction to proceed. Lowering the activation energy allows more molecules to reach the transition state and proceed.

enyzmes do not change the free energy of the reaction or the reaction equilibrium (∆G)

Question 5

Things that effect enzyme activity and rate

Answer 5

temperature- increases rate/activity because more collision between E and S (if temp too high will denature it)

pH- can alter the ionization state of the substrate or enzyme catalytic groups and if too high, it will denature the protein

Question 6

Catalytic strategies

Answer 6

**proximity **(inc effective concentration and correct orientation of substrate A w/ B)

**transition state stabilization **(oxyanion hole)

**covalent catalysis or nucleophilic catalysis **(serine binds to acyl group)

**general acid-base catalysis **(creates potent nucleophile serine)

metal ion catalysis

Question 7

Chymotrypsin protease action in steps

step 1

Answer 7

CATALYTIC TRIAD: aspartate-histidine-serine

aspartate COO- binds to H-N on one side of histidine changing its pKa

histidine N (general base) on other side pulls the H off of Serine’s hydroxyl group creating an alkoxide ion (acid-base catalysis)

Question 8

chymotrypsin protease action in steps

step 2

Answer 8

potent nucleophile seriene attacks the carbonyl bond of the amide forming a tetrahedral intermediate **which is stabilized in the oxyanion hole by hydrogen bonds on the enzyme (covalent catalysis)

*this tight binding is only possible in the transition state

Question 9

chymotrypsin action in steps

step 3

Answer 9

HisH+ donates a proton (general acid) to the amine portion which falls off and the acyl enzyme is created between the oxygen of serience and the carbonyl portion of the peptide

Question 10

chymotrypsin action in steps

step 4

Answer 10

water hydrolizes the bond and the carboxylic acid leaves

Question 11

How do endergonic reactions proceed?

Answer 11

∆G > 0 so not spontaneous

(products have more energy than reactants)

so you can drive it forward by using ATP or coupling it to an exergonic reaction

example) couple ATp hydrolysis (∆G = -30.5 kJ/mol) with glucose phosphorylation (∆G = 13.8 kJ/mol)

energies are additive: ∆G = -16.7 kJ/mol is still spontaneous

Question 12

Enzyme Kinetics: v versus E and v versus S

Answer 12

enzymatic rate increases linearly with enzyme concentraion

enzyme increases asymptotivally with increasing substrate concentration (reach a v max because saturated ES complex)

Question 13

Enzyme kinetics: Michaelis Menten Equation

Answer 13

v = Vmax [S] / Km + [S]

where:

v = initial velocity at a given substrate concentration [S]

Km is a contant (how tightly S binds to E, the tighter the binding, the smaller the Km and the less S needs to be used)

kcat x [E] = Vmax (higher the kcat, the faster the reaction)

S where Vmax/2 = Km (x intercept on hyperbolic plot)

Question 14

Enzyme Kinetics: Lineweaver-Burk plot

Answer 14

1/v = Km/Vmax[S] + 1/Vmax

in y = mx + b form

x intercept: -1/Km

y intercept: 1/Vmax

Question 15

What is Km?

Answer 15

units: M, mM, uM

it is a measure of the affinity of the substrate for the enzyme (i.ehow tightly do they bind…the more interactions between E and S there are, the lower the Km)

each enzyme has their own Km

constant but does change with pH and temperature

Question 16

Vmax and kcat

Answer 16

Vmax: everything is ES, maximum velocity with which the enzyme can catalyze the reaction

kcat: Vmax/[E]. Turnover number of an enzyme reflecting the number of moles of substrates converted to products per second per mol of enzyme

kcat is constant and only changes with pH and temperature

Question 17

(Brief) How enzymes can be regulated

Answer 17

extracellular signal –> TF –> protein

(mRNA can be degraded or protein can be degraded)

Enzyme can combine with a regulatory protein or be turned on and off with PO42- (covalent modification of AA)

Enzyme can bind to a ligand acting as an allosteric effector

Enzyme can be sequestered in subcellular organelle

Question 18

Enzyme phosphorylation and dephosphorylation

Answer 18

phosphorylated via protein kinase, makes very negative and changes catalytic activity

dephosphorylated via protein phosphatase

reversible reactions

Question 19

Enzymes regulated by specific proteolysis

Answer 19

irreversible way to activate enzyme

ex) digestive system, blood clotting, insulin, collagen, apoptosis via caspases
ex) pancreatic zymogens (pepsinogen cleaved to form pepsin* which is now active)

Question 20

Enzyme Inhibition

irreversible

Answer 20

molecules covalently bind to the enzyme to inhibit activity

these inhibitor molecules look like the natural substrate

ex) penicillin bonds with transpeptidase preventing the synthesis of bacterial cell wall

Question 21

Enzyme Inhibition

reversible inhibition

Answer 21

1) competitive: binds to same active site as substrate. looks like the substrate but doesnt react
2) noncompetitive: binds to a separate sitre from the active site and causes a conformational change in the enzyme at the active site

Question 22

Competitive vs Noncompetitive inhibitors

kinetics that distinguish them

Answer 22

competitive inhibitor: Km increases (bc doesnt let substrate bind to the enzyme so Km increases), Vmax doesnt change because a high [S] overcomes inhibition

noncompetitive inhibitor: Km doesn’t change but Vmax decreases (bc you changed the catalytic activity of the enzyme)

High [S] doesnt overcome inhibition

Question 23

Competitive vs Noncompetitive Inhibition

How do you distinguish them on a LB graph?

Answer 23

competitive inhibitor: enzyme in the presence of inhibitor will show the same intercept on the y axis as the regular enzyme but will have a smaller x intercept because it is the reciprocal

noncompetitive inhibitor: enzyme in the presence of inhibitor will have a different y intercept than the regular enzyme (it will be higher because it is reciprocal) and they will converge at the same x intercept

Question 24

How can you make a POTENT competitive inhibitor? i.e. an inhibitor that is really good at its job

Answer 24

TRANSITION STATE ANALOG

why? here it is stable

recall- enzymes bind more tightly to the transition state substrate than the regular substate.

as a result. the enzyme will bind really tight to the transition state analog competitive inhibitor

Question 25

Feedback Regulation (involving enzymes and products)

Answer 25

metabolic pathways are regulated

for example) enzyme 1 in the pathway may be allosteric and two much product E downstream will bind to enzyme 1 and inhibit it (stopping the entire pathway)

or, enzyme T further down in the may activate enzyme 1 and effect it in this manner

Question 26

Allosteric enzymes

properties

Answer 26

these enzymes have an active site and an allosteric site

they are oligomeric (i.e. at least a dimer or some sort like Hemoglobin)

active site binds S and the allosteric site binds the effector or allosteric molecules that regulate enzyme activity (allosteric molecules don’t look like substrate)

Question 27

Allosteric Enzymes

How can they be kinetically distinguished?

Answer 27

does not show a hyperbolic v versus [S] curve

instead show a sigmoidal curve

why? cooperative substrate binding meaning that the binding of the substrate at one point on the molecule alters the binding of the substrate at another part of the molecule

ex) hemoglobin

Question 28

Allosteric Enzymes

mechanism of regulation

Answer 28

they exist in 2 confirmations: active R-state (relaxed) and inactive T-state (tense)

R has a high affinity for S (T is opposite)

why? substrate binding stabilizes the molecule but there are many active sites. It is difficult for the first S to bind to the T state. Once the 1st S binds, the molecule opens up and all substrates can bind to an active site

Question 29

Allosteric regulators/effectors modulate the T to R equilibrium

Answer 29

allosteric activators- stabilizes the R-state which increases S binding (which can increase activity)

allosteric inhibitors- stabilizes the inactive T state which decreases S binding (which can decrease activity)

Question 30

Allosteric Inhibitors

K type and V type

Answer 30

K type: increases the Km (makes substrate binding weaker) but does not affect Vmax

at low [S], the activity of enzyme is lower than normal and at high [S], activity of the enzyme is normal

V type: decreases the Vmax but will not affect the Km. The affinity of S for E is not affected.

At all [S], the activity of E is lower

Question 31

Allosteric activators

K type and V type

Answer 31

K type: decreases Km (increasing strength of S to E bond), will not effect Vmax

at low [S], activity of E is higher and at high [S], activity of E is normal (saturated ES complex)

V type: increases V max and doesn’t effect Km

at all [S], the activity of E is higher

Question 32

What type of allosteric inhibition is this?

Answer 32

K type

Question 33

What type of allosteric inhibition is this?

Answer 33

V type

Question 34

What type of allosteric activation is this?

Answer 34

K type

Question 35

What type of allosteric activation is this?

Answer 35

V type

Question 36

Enzymatic cofactors

Nomenclature

Answer 36

apoenzyme + cofactor (or coenzyme) = holoenzyme

holoenzyme- active enyme with non protein component

without active component? aponzyme (a means without)

cofactor- metal ion i.e. Zn or Fe

coenzyme- small organic molecule that can be a) cosubstrate if transient (NAD+) or prosthetic group if permanent (heme)