10/31 Enzymes (Not Finished)

Front 1

Thermodynamics are about

Back 1

how stable things are.

Front 2

Kinetics are about

Back 2

speed, rate, velocity.

Front 3

What are enzymes?
What powers do they have (2)?
What are they made of?

Back 3

catalysts
catalytic power and specificity
proteins or RNA or maybe other things I guess

Front 4

What is a catalyst?

Back 4

Substance that increases the rate of a rxn but is in the same state before and after the rxn

Front 5

An enzyme reacts ________ with substrate S to form an enzyme-substrate complex ES.

The ES complex then _____________ forms the product P and ____________ the enzyme catalyst E

Back 5

reversibly

irreversibly
regenerates

Front 6

Enzymes are _____ specific in their choice of ______ (_____).

Wasteful side rxns are ______

Back 6

highly reactants (substrates)

rare

Front 7

What enzyme cleaves peptide bonds and by what rxn?

What two examples of specificity did they give?

Back 7

Proteases or proteolytic enzymes by hydrolysis (uses water)

Trypsin cuts next to K/R and any other.
Thrombin cuts between Arg and Gly

Front 8

What do you call the bond that proteolytic enzymes break?

Back 8

Scissile bond between COO and NH of two adjacent amino acids.

Front 9

What interactions does an enzyme use to ID a substrate?

Back 9

Van der Waals, electrostatic, hydrogen bonding, hydrophobic interactions

Front 10

What are cofactors?

What are enzymes called without their cofactor?
With?
What are the two groups of cofactors?

Back 10

Small molecules bound to the enzyme

apoenzymes
holoenzymes
metals and small organic molecules (coenzymes).

Front 11

Coenzymes can be utilized in two ways:

What are often precursors to essential coenzymes?

Back 11

tightly bound as prosthetic group or bound and released like substrates

Vitamins (though they're not all necessarily used as coenzymes: fat-soluble vitamins do other things sometimes)

Front 12

4 rxn classifications for organic chemistry:

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rearrangement A -> C
elimination A -> C + D
addition A + B -> C
substitution A + B -> C + D

Front 13

4 rxn classifications for biochemistry because they're so cool

Back 13

isomerase/mutase A -> C
lyase A -> C + D
ligase A + B -> C
oxidoreductase/transferase/hydrolase A + B -> C + D

Front 14

Oxidoreductase class rxn

Often called _______

Back 14

promote oxidation-reduction reactions (taking off protons/oxidizing things, NAD+/NADH)

dehydrogenases

Front 15

Transferase example

Back 15

transfer rxn.

ATP + glucose -> G6P + ADP

Front 16

Hydrolases

Enzyme class

Back 16

catalyze hydrolysis- adding parts of water to molecule to split

Proteases

Front 17

What do enzymes do theoretically to catalyze rxns?

What is the implication here?

Back 17

Help substrate get through transition state (S‡) easier by lowering the free energy of activation (∆G‡ ) as the substrate transitions to lower energy product.

Enzymes will only catalyze rxns that will happen anyway (product is at lower G i.e. rxn has -∆G)

Front 18

How do enzymes catalyze?

Why is this important for kinetics?

Back 18

Bind transition states S‡ which lowers ∆G‡

The velocity (V) of rxn depends on ∆G‡

Front 19

Formation of the _____-_______ complex: E + S <-> ES

The __________ are bound in the _____ _____ of enzymes.

Back 19

enzyme-substrate
substrates active sites

Front 20

Rxn velocity is dependent variable of

Back 20

concentration of substrate

Front 21

Description of typical active sites

Back 21

Contains the catalytic residues
Tend to be a small part of the enzyme
3D, often clefts or crevices

Front 22

How all do active sites keep specificity?

Back 22

Have multiple weak interactions with substrates to bind (h bonds, van der Waals, etc)

Use geometry of active site (hydrophobic pockets, anion hole)

Front 23

What are the two active site geometry models?

Back 23

Lock and key model. Substrate fits exactly.
Induced fit model: Kind of close to substrate form but not exactly. Puts it in the orientation you want.

Front 24

Enzyme kinetics: V

Back 24

Number of mols of product formed per second

Front 25

Enzyme kinetics: K_m_ (2 definitions)

Equation using constants

Back 25

concentration of S at which rxn rate is half the maximal rate (V_max_); [S] where have of E active sites are occupied

Km = k-1 + k2 / k1 = rate of ES breakdown/rate of ES formation

Front 26

What are the rules of making enzyme kinetic graph?

Back 26

Fixed total concentration of enzyme [E_T_]

Front 27

_________-___________ Model of Enzyme Kinetics

E + S <k1 and k-1> ES --k2-> E + P
Two assumptions.
What is V?

Back 27

Michaelis-Menten Model

k and k-1 are not necessarily equal
Assumes P will not go backwards to S

V = k_2_[ES]

Front 28

Michaelis-Menten Model
E + S <k1 and k-1> ES --k2-> E + P

Rate of formation of ES?
Rate of breakdown of ES?
What is steady state rate?

Back 28

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

At steady state [ES] remains constant, [S] and [P] changing

Front 29

Vmax

Back 29

Maximal rate. Happens when [S] >> Km.

Front 30

Michaelis-Menten equation

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

What two conclusions do we draw from this?

Back 30

Higher the [S] the closer V is to Vmax

Km is equal to [S] at which V = Vmax

Front 31

What's the straight line version of Michaelis-Menten?

Back 31

Lineweaver-Burk Plot

Front 32

What does Lineweaver-Burk Plot show?

What can you infer when comparing different rxns?

Back 32

slope = Km/Vmax
y Intercept is 1/Vmax
x intercept is -1/Km

The type of inhibition based on changes in intercepts

Front 33

Km is really a measure/reflection of what?

High/low values of Km reflect what?

What does Km values mean for speed of the enzyme?

Back 33

How well the substrate binds. It's the dissociation constant for the breakdown of ES complex

High value shows weak binding (high k-1)

Not much. Not directly related to kinetics.

Front 34

If k-1 >> k2 then Km is a measure of

Back 34

strength of ES complex

Front 35

Vmax defined twice

Back 35

Maximal catalytic rate

All enzymes have filled active site

Front 36

Turnover number

How can you use it in equation?

Back 36

Number of S converted to P /second when enzyme is fully saturated

It's a constant for a given enzyme (k2)

Vmax = k2[Et]

Front 37

Multiple substrates
A + B <-> P + Q

Usually the transfer of a _________ ______.
In an oxidation-reduction rxt, ____ are transferred.

Two classes:

Back 37

functional group
electrons

sequential displacement and double displacement

Front 38

In a sequential displacement rxn...

In a bisubstrate rxn, a _________ complex is formed

What are the two sub classes of this reaction?

Back 38

all substrates must bind enzyme before rxn starts

ternary

Sequential ordered and Random sequential

Front 39

Sequential displacement: Sequential ordered

Example

Back 39

Order of substrate binding and product release matters (all must be present before rxn)

lactate dehydrogenase
NADH then Pyruvate, rxn, then let go lactate then NAD+

Front 40

Sequential displacement: Random sequential

What characterizes these enzymes?

Example

Back 40

Doesn't matter which substrate binds first or which produce is released first (all must be present before rxn)

Tend to have more open active sites.

So creatine kinase + ATP to phosphocreatine + ADP

Front 41

Double displacement mechanism type (and another name)

In these rxns there is a _______ _______ intermediate.

Back 41

ping-pong rxn
E + S1 -> -P1 and E+group-> + S2 -> P2 + E
(A functional group is transferred to the enzyme to pass on)

substituted enzyme

Front 42

_______ enzymes do not follow Michaelis-Menten kinetics.

What kind of curve on kinetics chart?

Back 42

Allosteric

sigmoidal

Front 43

What inhibits enzyme?

Two classes of inhibition

Back 43

Natural biological regulation but also drugs and toxic agents.

Reversible or irreversible (likely covalent, inhibitor doesn't come off)

Front 44

Two types of reversible inhibition and general idea of them.

Back 44

Competitive (usually higher affinity than substrate) and noncompetitive (non active site interaction that makes enzyme have lesser affinity for substrate)

Front 45

How do the two reversible inhibitor types affect kinetics of rxn?

Back 45

Competitive: Vmax unchanged, Km increased
Can overcome by increasing [S]

Noncompetitive: Decreases Vmax, Km unchanged
can't overcome with [S]

Front 46

Reading Lineweaver plot what to focus on to tell inhibition type?

Back 46

x intercept for Km
y intercept for Vmax

Front 47

What makes a good inhibitor?

Example

Back 47

transition state analog

Proline racemase inhibited by planar molecule that looks like transition state

Front 48

IC_50_

Back 48

effectiveness of drug is usually given as IC_50_

An IC_50_ is concentration of inhibitor/drug at which half the maximal enzyme activity.

It's unfortunative because IC50 depends on situation in which it's measured

Front 49

3 classes of irreversible inhibition

Back 49

group-specific - bind to specific side chains

affinity labels - structurally similar ot substrate but then covalently modify active site

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Front 51

Suicide inhibitors

Back 51

mechanism-based inhibitor

bind as substrate and normal catalytic activity starts but then the inhibitor produces a reactive species that covalently binds to the enzyme

Front 52

how does penicillin work?

Back 52

irreversible inhibition: suicide inhibitor. blocks bacterial cell wall synthesis.

gram positive bacteria has peptidoglycan wall

does it with glycopeptide transpeptidase. Looks like two Ala peptides. It binds to a Ser as per normal but can't continue

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Front 55

4 strategies for enzymes
-active site contains reactive group that's temporarily covalently modified
-a molecule/residue plays role of proton donor/acceptor
-uses a bound metal ion
two substrates brought together on single binding surface to enhance rate rxn

Back 55

covalent catalysis
general acid-base catalysis
metal-ion catalysis
catalysis by approximation

Front 56

hwo do proteases work?

Back 56

catalyze by hydrolysis.
Important for digestion.

Front 57

Chymotrypsin

Back 57

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Front 58

Catalytic triad of chymotrypsin

Back 58

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Front 60

protease: chymotrypsin catalytic mechanism

Back 60

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Front 61

oxyanion hole

Back 61

helps stabilize the very unstable tetrahedral intermediates

Front 62

How does chymostrypsin have specificity?

Back 62

has a large hydrophobic cavity called the S1 pocket next to the active site

Front 63

lots of serine proteases but

Back 63

they have different specificities

trypsin and elastase are homologs or paralogs to chymotrypsin

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Front 65

Serine proteases that are not homologous to chymotrypsin

Back 65

subtilisin
carboxypeptidase II

Front 66

other classes of proteases (besides serine)

Back 66

cysteine proteases: use cysteine instead
aspartyl proteases: two Asp groups plus water
metalloproteases: bind metal ion (Zn commonly) that activates water to attack peptide bonds

Front 67

Because proteases are important they are targeted like

Back 67

HIV-1 protease inhibition by crixivan

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carbonic anhydrases. mutations in this causes

Back 71

mental retardation or osteropetrosis (excessive bone density plus anemia)

Front 72

Carbonic anhydrase: discovered early. found

Back 72

had bound zinc that associated with water. have 7 genes of these

water becomes OH- with electron from other H so

speeds rxn 10^6

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