Week 6: M-M Inhibitors, Allosteric Enzymes, & Control of Activity

Question 1

Mixed noncompetitive inhibition is similar to noncompetitive inhibition but binding of inhibitor DOES affect _________

and still lowers ______

Answer 1

binding of substrate and vice versa

Vmax

Question 2

Similar to a Lineweaver-Burk plot for a noncompetitive inhibitor, one for a mixed noncompetitive inhibitor:

_____ and _______ change
however, _______ changes from that of noncompetitive because _______

Answer 2

slope, y-intercept

x-intercept because -1/KM becomes a smaller negative value

Question 3

Mixed competitive inhibition:
______ decreases
_______ increases (or decreases)

Answer 3

Vmax
KM

Question 4

With pure noncompetitive inhibition, the binding of the inhibitor does not change __________ at all and vice versa; thus the _______ does not change. With mixed inhibition, the _______ such that the ______ for the substrate is ______ in the presence of inhibitor.

Answer 4

affinity of the enzyme for substrate, KM

substrate and inhibitor do affect each other, KM

different

Question 5

In uncompetitive inhibition, inhibitor can only bind to the _______, never the _______

Answer 5

ES complex
free enzyme

Question 6

In uncompetitive inhibition, ______ decreases because it causes a ______ of product formation.

KM _______ because Le Chatliers Principle: as ______ formed, formation of more ______ favored.

However, because ______ decreases remember it still lowers enzyme activity.

Answer 6

Vmax, lower rate

decreases, EIS, ES

Vmax

Question 7

Lineweaver-Burk equation:

Answer 7

1/V = KM/Vmax x 1/[S] + 1/Vmax

Question 8

Lineweaver-Burk plot for uncompetitive plot contains ________ meaning ______ slope, ______ intercepts

Vmax and KM _______ to same extent

Answer 8

parallel lines
same slope
different intercepts

decrease

Question 9

Lineweaver-Burk equation for uncompetitive inhibition:

Answer 9

1/V = a’KM/a’Vmax x 1/[S] + a’/Vmax

1/V = KM/Vmax x 1/[S] + a’/Vmax

Question 10

Irreversible inhibition is _______ binding of inhibitor to enzyme, causing _______ inactivation

Answer 10

covalent
permanent

Question 11

Suicide substrates: Molecules bind to enzyme irreversibly and ______ it, reaction can’t move ________ and can’t ________ either

also called _______ substrates

Answer 11

inactivate, through the reaction sequence, reverse it

Trojan horse (sneak in)

Question 12

Allosteric enzymes can be controlled by many different mechanisms, including inhibition and activation by ________.

Answer 12

reversibly binding molecules

Question 13

_________ is a common way to regulate an allosteric enzyme that is part of a complicated pathway.

Answer 13

Feedback inhibition

Question 14

What features distinguish enzymes that undergo allosteric control from those that obey the Michaelis–Menten equation?

Allosteric enzymes display ________ kinetics when rates are plotted versus substrate concentration. Michaelis–Menten enzymes exhibit _______ kinetics. Allosteric enzymes usually have _________, and the binding of substrates or effector molecules to one subunit __________.

Answer 14

sigmoidal

hyperbolic

multiple subunits

changes the binding behavior of the other subunits.

Question 15

ATP acts as a _______ of ATCase, and CTP acts as ________.

Answer 15

positive effector

an inhibitor

Question 16

_____ acts as a positive effector of ATCase, and _____ acts as an inhibitor.

Answer 16

ATP, CTP

Question 17

The sigmoidal curve of the plot of reaction rate against substrate concentration reflects the _________ of allosteric enzymes

Answer 17

cooperative behavior

Enzymes that exhibit cooperativity do not show hyperbolic curves of rate versus substrate concentration. Their curves are sigmoidal. The level of cooperativity can be seen by the shape of the sigmoidal curve.

Question 18

Inhibitors make the shape of the curve ______ sigmoidal, thus ______ cooperative

Answer 18

more, more

Question 19

Activators make the shape of the curve _____ sigmoidal, thus _____ cooperative

Answer 19

less, less

Question 20

Allosteric proteins (including enzymes) are ones in which ________ at one site affect the _______
* Most common in proteins with _________
* Can have positive or negative _______

Answer 20

subtle changes
structure and function at another site

quaternary structure

positive or negative cooperativity

Question 21

Structural organization of enzyme ATCase:

Answer 21

2 catalytic trimers + 3 regulatory dimers

Question 22

Regulatory subunits are where the ________

Answer 22

allosteric effectors bind (inhibitors and activators)

Question 23

Catalysis occurs in absence of ________
* No ______ in that case

Answer 23

R subunits
regulation

Question 24

Why is ATCase not considered a “competitive” inhibitor even though for both this and competitive inhibitors, Vmax stays constant?

Answer 24

Doesn’t follow lock-and-key induced fit and M-M kinetics hyperbolic plot associated with “competitive” inhibitor (instead sigmoidal)

Question 25

K systems:

Answer 25

Inhibitors/activators change K values (substrate affinity)

Question 26

K0.5:

Answer 26

Substrate level at ½ Vmax in a K system
* Analogous to KM for nonallosteric enzymes

Question 27

V systems:

Answer 27

Inhibitors/activators that change Vmax

Question 28

Allosteric effector: ______, _______, or ________
that binds to _______ enzyme and affects activity

Answer 28

Substrate, inhibitor, activator
allosteric

Question 29

Homotropic effects: Occur when several ______
molecules are bound
* Ex:

Answer 29

identical

Binding of aspartate to ATCase

Question 30

Heterotropic effects: Occur when _____
substances are bound
* Ex:

Answer 30

different

Activation of ATCase by ATP when substrate also bound

Question 31

Michaelis-Menten enzyme never involves product ______ and product production kinetics follows a ______ saturating V vs. [S] curve.

Answer 31

feedback activation
hyperbolic

Question 32

ATP, a purine nucleotide, is a _______ of ATCase. This ______ action of ATP is thought to be due to the cell’s need to keep ________ approximately balanced.

Answer 32

positive regulator
positive feedback
purine and pyrimidine nucleotide levels

Question 33

A K system is an ______ enzyme for which the _______ of the enzyme for its substrate is altered in the presence of effector and for which the apparent ______ does not change.

Answer 33

allosteric, apparent affinity

Vmax

Question 34

Both _______ cooperativity cause the V vs. [S] plot to deviate from the ______ plot of a Michaelis-Menten enzyme.
The ________ is reflected in the extent of curvature of the V vs. [S] plot.

Answer 34

positive and negative
hyperbolic

degree of cooperativity

Question 35

Triaclyglycerols are typically used for the ______; they do not appear in ______ of animals. They are mostly deposited in _______ and are also found in the ______ in the form of very low density lipoprotein (VLDL) and chylomicrons.

Answer 35

storage of fatty acids
cell membranes
adipose tissue
blood

Question 36

Waxes are found on the ______ of leaves.

Waxes frequently form ______ for plants as well as animals.

Answer 36

outside
protective coatings

Question 37

In concerted model, all enzyme subunits are in the _____ form, either R or T. Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeaux proposed this model.

Answer 37

same

Question 38

In sequential model, enzyme subunits can be ________ independent of each other.

Answer 38

in either the R or the T form

Question 39

What property of a metal ion makes it a useful cofactor in enzyme-mediated catalysis?

Answer 39

A metal ion can act as a Lewis acid.

A metal with an excess complement of electrons can readily donate a lone pair of electrons, forming a metal ion. Metal ions are electron deficient and can act as Lewis acids, accepting electrons into unoccupied d-orbitals to form metal complexes.

The donation of electrons from a Lewis base (the substrate in the case of enzyme-mediated catalysis) can cause electronic strain on other bonds in the substrate, weakening them and making them easier to break.

Question 40

R:X + Z: –> R:Z + X:

SN1 reactions are _______ in nature. In the illustrated reaction, R:X is the molecule that breaks down to control the rate of the reaction. Only later does X: react with Z:. Thus, this reaction will have a first order rate constant and its rate will depend on [R:X].

SN2 reactions are ______ in nature. In the illustrated reaction, R:X will react with Z: initially to form R:Z. Thus, this reaction will have a second order rate constant and its rate will depend on both [R:X] and [Z:].

Answer 40

unimolecular

bimolecular

Question 41

Lipids can be grouped based on:

having a preponderance of ______ groups.
their _______ in nonpolar solvents.

Answer 41

nonpolar
high solubility

Question 42

A K system is an ______ enzyme in which the binding of inhibitor alters the apparent _______ needed to reach one-half Vmax, S0.5.

Answer 42

allosteric, substrate concentration

Question 43

A V system is an allosteric enzyme in which the binding of inhibitor changes the _____ of the enzyme but not the _____ .

Answer 43

Vmax, S0.5

Question 44

S0.5 is the substrate concentration that leads to _____ the maximal velocity. This term is used with _____ enzymes, where the term _____ is not appropriate.

Answer 44

half of

allosteric

KM

Question 45

In the concerted model for allosteric behavior, the binding of substrate, inhibitor, or activator to one subunit ________ between an active form of the enzyme, which binds substrate strongly, and an inactive form, which does not bind substrate strongly.

Answer 45

shifts the equilibrium

Question 46

In the sequential model, the binding of substrate induces the __________ in one subunit, and the change is ______ along to other subunits.

Answer 46

conformational change

subsequently passed

Question 47

Allosteric enzymes tend to be enzymes with ______ structure

Answer 47

quaternary

Question 48

Since the T form is much _____ to bind substrate, its dissociation constant ([E][S]/[ES]) would be _____

Answer 48

less likely
very large

Question 49

Enzyme mostly in ______ in the absence of substrate

Answer 49

T form

Question 50

In concerted model, change from T to R (conformations) change _______.

Protein has two conformations:
* Active R (relaxed) - Binds substrate with ______
* Inactive T (tight or taut) - Binds substrate with _______

Answer 50

simultaneously
high affinity
lower affinity

Question 51

L =

Answer 51

equilibrium ratio of T:R
[T]&raquo_space; [R]

Question 52

c =

Answer 52

ratio of KR:KT

Question 53

Presence of substrate shifts equilibrium from _______ to _____
* Driven by LeChâtlier’s principle
* Substrate binds ______

Answer 53

T (inactive)
R (active) form

R form

Question 54

Higher L means higher favorability of ______ and ______ sigmoidicity

Answer 54

free T form (since L = T/R)
greater

Question 55

Lower c means higher affinity of ______, meaning ______ sigmoidicity

Answer 55

R form for S
greater

Question 56

Higher c means _____ sigmoidicity

Answer 56

less

Question 57

Binding of activator (A) shifts equilibrium to favor ____
* Less need for _____
* Curve ______ sigmoidal (________- cooperativity)

Answer 57

R
S to shift to R form
less sigmoidal (reduced cooperativity)

Question 58

Binding of inhibitor (I) shifts equilibrium to favor ____
* ______ need for S to shift to R form
* Curve _____ sigmoidal (______ cooperativity)

Answer 58

T
Greater
more
increased

Question 59

When c = 0, ____ is infinite because the substrate ______. Cooperativity in this case is ______.

As c increases, it means the difference in binding for T and R ______. We see line become ______ sigmoidal as the need for cooperativity ______.

Answer 59

KT is infinite
does not bind to KT
high (once some substrate has bound, more is likely to)

decreases
less
drops

Question 60

In sequential model, binding of ____ induces ______ from T to R form

Answer 60

S
conformational change

Question 61

In sequential model, substrate binds ____ and changes to ______,

induces same change to ______, binds substrate with higher _____

Answer 61

T form
R form

adjacent subunit
affinity

Question 62

Sequential Model
* Cooperative binding of S to allosteric enzyme
* _____ is favored by allosteric activator
* Allosteric inhibitor binds ____
* Conformational change to S binding site
* Allosteric inhibition occurs by induced-fit mechanism

Answer 62

R form
T form

Question 63

Sequential model has a unique feature: Negative cooperativity –>

Answer 63

Negative cooperativity: Binding of first ligand results in lower affinity for next

Question 64

In the concerted model, all the subunits in an allosteric enzyme are found _____. They are in equilibrium, with each enzyme having a characteristic ratio of the T/R. In the sequential model, the subunits _____.

Answer 64

the same form, either the T form or the R form

change individually from T to R

Question 65

The _____ model can explain negative cooperativity, because a substrate binding to the T form could induce other subunits to switch to the T form, thereby reducing ______.

Answer 65

sequential

binding affinity

Question 66

The L value is the ______ ratio of the ____. The c value is the ratio of the _______ for substrate and the two forms of enzyme, such that _____.

Answer 66

equilibrium, T/R form

dissociation constants

c = KR/KT

Question 67

Phosphorylation is the _____ attachment of a _____ group to an amino acid ______

Answer 67

covalent
phosphoryl
side chain

Question 68

control of enzyme activity through phosphorylation: usually phosphorylated on ______ groups of ________

Answer 68

hydroxyl
Ser, Thr, Tyr

Question 69

Phosphoryl group usually comes from _____

Answer 69

ATP

Question 70

_____ add phosphoryl groups and ______ take them off

Answer 70

Kinases
phosphatases

Question 71

Glycogen phosphorylase:
Phosphorylase _____ more
active than ______
* Two forms (a & b) respond
to different ______

Answer 71

a, b form

allosteric
effectors

Question 72

A kinase is an ______ that ______ a protein using a high-energy phosphate, such as _______, as the phosphate donor.

Answer 72

enzyme
phosphorylates
ATP

Question 73

______ are the three most often phosphorylated amino acids in proteins that are acted upon by kinases. ______ is another one that is often phosphorylated.

Answer 73

Serine, threonine, and tyrosine
Aspartate

Question 74

Glycogen phosphorylase is controlled allosterically by several molecules. In the muscle, ______ is an allosteric activator. In the liver, ______ is an allosteric inhibitor. Glycogen phosphorylase also exists in a phosphorylated form and an unphosphorylated form, with the _________ being more active.

Answer 74

AMP, glucose

phosphorylated form

Question 75

Inactive nascent proteins are called _____:

Enzymes ____ and ready to go when _____, this contributes to an amplifying effect to give large effect in ________

Answer 75

zymogens
pre-formed, activated
small amount of time (faster responses)

Question 76

General structure of chymotrypsinogen:

Answer 76

cross-linked by disulfide bonds

Question 77

Chymotrypsinogen is first cleaved by _____ to give one active form, cleaves itself to give most ______

Part of _____ removed, which alters ______ to give _____

Answer 77

trypsin
most active form

primary structure
tertiary structure
active form

Question 78

Why is it necessary or advantageous for the body to make zymogens?

Zymogens are often seen with ______ that are produced in one tissue and used in another. If the enzyme were active immediately upon production, it would _____, where it would cause _____. By having it produced as a zymogen, it can be ______ to the digestive tissue, such as the stomach or small intestine, where it can then be activated.

Answer 78

digestive enzymes
digest other cell proteins
great damage

safely made and then transported

Question 79

Nature of active site. For each enzyme you would have to consider:

Answer 79

which amino acid side chains (residues) are present

spatial relationship of essential amino acids in active site

chemical mechanism in which essential amino acid residues catalyze the reaction

Question 80

General reaction of chymotrypsin:
_____ substrate
Can use _______

Two phases:

Answer 80

natural
p-Nitrophenyl acetate

p-nitrophenyl acetate hydrolyzed by chymotrypsin in two stages:
* Initial burst
* Formation of covalent intermediate, rapid
* Slower phase
* Hydrolysis
* Release of acetate

Question 81

3 residues of the catalytic triad:

Answer 81

Ser, His, Asp

Folding of backbone positions essential amino acid residues around active-site pocket

Question 82

Enzymes that catalyze biochemical reactions have been formally classified into ______ subgroups according to reaction type.

Answer 82

six

Question 83

6 classes of enzymes:

Answer 83

Oxidoreductase
Transferase
Hydrolase
Lyase
Ligase
Ismoerase

Question 84

Oxidoreductase

Answer 84

facilitates redox reactions

Question 85

Transferase

Answer 85

catalyze the transfer or exchange of certain groups

Question 86

Hydrolase

Answer 86

adding water to break bonds

Question 87

Lyase

Answer 87

breaking or making bonds

Question 88

Isomerase

Answer 88

moving the order of attachment
A-B-C –> A-C-B

Question 89

Ligase

Answer 89

ATP hydrolyzed to release energy

Question 90

Many enzymes require _____ for activity

Answer 90

cofactors

Question 91

Cofactors:
Coenzymes:

Answer 91

coenzymes, metal ions
prosthetic group, cosubstrate

Question 92

NAD+ is an example of a ______
Participate in _____ reactions

Answer 92

cosubstrate
redox

Question 93

Structural components of NAD+?

Answer 93

Nicotinamide ring, adenine ring, two sugar-phosphates

Question 94

Where prosthetic groups are ______ to enzyme, cosubstrates can _______

Answer 94

bound
enter or leave active site

Question 95

apoenzyme

Answer 95

before cofactor added, not functional

Question 96

B6 vitamins participate in amino acid ______

Answer 96

biosynthesis

Question 97

_______ are the two most critical amino acids in the active site of chymotrypsin.

Answer 97

Serine and histidine

Question 98

Why does the enzyme reaction for chymotrypsin proceed in two phases?

The initial phase releases the _____ and involves an _____ intermediate. This step is _____ than the second part, in which _____ comes into the active site and breaks the______ bond.

Answer 98

first product, acylenzyme

faster

water, acyl–enzyme

Question 99

Briefly describe the role of nucleophilic catalysis in the mechanism of the chymotrypsin reaction.

In the first step of the reaction, the _____ is the nucleophile that attacks the substrate peptide bond. In the second step, ______ is the nucleophile that attacks the acyl-enzyme intermediate.

Answer 99

serine hydroxyl

water

Question 100

Explain the function of histidine 57 in the mechanism of chymotrypsin.

Answer 100

Histidine 57 performs a series of steps involving general base catalysis followed by general acid catalysis. In the first phase, it takes a hydrogen from serine 195 , acting as a general base. This is followed immediately by an acid catalysis step, in which it gives the hydrogen to the amide group of the peptide bond that is breaking. A similar scheme takes place in the second phase of the reaction.

Question 101

Explain why the second phase of the chymotrypsin mechanism is slower than the first phase.

Answer 101

The first phase is faster for several reasons. The serine at position 195 is a strong nucleophile for the initial nucleophilic attack. It then forms an acyl-enzyme intermediate. In the second phase, water is the nucleophile, and it takes time for water to diffuse to the right spot to perform its nucleophilic attack. It is also not as strong a nucleophile as the serine. Therefore, it takes longer for water to perform its nucleophilic attack and break the acyl-enzyme intermediate than it takes for serine to create it.

Question 102

Explain how the pKa for histidine 57 is important to its role in the mechanism of chymotrypsin action.

Answer 102

Histidine 57 exists in both the protonated and unprotonated form during the chymotrypsin reaction. Its pKa of 6.0 makes this possible in the physiological pH range.

Question 103

Common organic reaction mechanisms, such as ______ and ______, are known to play roles in enzymatic catalysis

Answer 103

nucleophilic substitution

general acid–base catalysis

Question 104

In biochemistry mechanisms, what group is often attacked by a nucleopile?

Answer 104

The carbon of a carbonyl group is often attacked by a nucleophile.

Question 105

General acid catalysis is the part of an enzyme mechanism in which an amino acid or other molecule donates a _______ to another molecule.

Answer 105

hydrogen ion

Question 106

Absolute specificity – Catalyzes reaction of ______ to specific product

Answer 106

one unique substrate

Question 107

Relative specificity – Catalyzes reaction of _______ to give structurally related products

Answer 107

structurally related substrates

Question 108

Stereospecificity –
Suggests ______ binding

Answer 108

One stereoisomer reacted or
formed in preference to all others

asymmetric binding

Question 109

Nature of transition state is ______
between substrate and product
* ______ from either substrate or product

Answer 109

intermediate in structure

Different shape

Question 110

Transition-state analog: Synthesized compounds
that ______ of transition state

Answer 110

mimic form

Question 111

Abzymes:
Antibodies with ______
Raised in response to ______

Answer 111

catalytic activity
transition state analogues

Question 112

What is the relationship between a transition-state analog and the induced-fit model of enzyme kinetics?

Answer 112

The induced-fit model assumes that the enzyme and substrate must both move and change to conform to each other perfectly. Thus, the true fit is not between the enzyme and substrate but between the enzyme and the transition state of the substrate on its way to product. A transition-state analogue fits the enzyme nicely in this model

Question 113

Explain how a researcher makes an abzyme. What is the purpose of an abzyme?

Answer 113

An abzyme is created by injecting a host animal with a transition-state analogue of a reaction of interest. The host animal makes antibodies to the foreign molecule, and these antibodies have specific binding points that mimic an enzyme surrounding a transition state. The purpose is to create an antibody with catalytic activity.

Question 114

Most coenzymes are derivatives of compounds we call _____.

Answer 114

vitamins

For example, nicotinamide adenine dinucleotide is produced from the B vitamin niacin. Flavin adenine dinucleotide comes from riboflavin.

Question 115

Proteins, nucleic acids, and carbohydrates are grouped by common structural features found within their group. What is the basis for grouping substances as lipids?

Answer 115

Solubility properties (insoluble in aqueous or polar solvents, soluble in nonpolar solvents). Some lipids are not at all structurally related.

Question 116

An allosteric enzyme will feature a ______ curve that represents _____, the changes that occur in the binding
activity of an enzyme in response to the binding of a ligand / substrate. This is possible because of the ______
present on the enzyme that will allow for regulation and multiple active sites.

Michaelis-Menten enzymes will always have a ______ curve because they are ______ structure proteins; they only
have one subunit that can bind one ligand at a time. While they do have sites for inhibitors to bind, they do not have
allosteric effectors that can alter binding patterns.

Answer 116

sigmoidal
cooperativity
multiple subunits

hyperbolic
tertiary

Question 117

The concerted model is an ‘all-or-nothing’ model that has substrates bind to the ______ of the enzyme. The equilibrium of
R/T form is determined by LeChatlier’s principle, where the presence of substrate will shift the enzyme to have all of its
subunits to be _____ where it can bind. This equilibrium is also affected by the presence of activators / inhibitors that can
‘override’ the presence / absence of substrate. Most molecules exist in the _____ when substrate is absent, which is also
explained by two equilibrium constants:

Answer 117

R form, R form
T form

L: Ratio of T state / R state. This is high in the concerted model (more T state)

C: Kr/Kt (dissociation constants). This value is low in the concerted model (more T state as Kr &laquo_space;Kt)

Question 118

The sequential model, on the other hand, has substrates bind to the ______ of the enzyme. This binding to the _______ will
shift the bound subunit to the _____ along with the ______, indicating that conformational changes happen ______, not all at once. Inhibitors will bind to the T form of the enzyme and induce conformational changes in adjacent
subunits to lower _______. The sequential model has the unique property of also allowing for the binding of one ligand to
______ the affinity of binding for the next one. This is called ________, which can only be explained by the
sequential model; the concerted model does not allow for such a feature

Answer 118

T form, T form
R form
adjacent subunit
one at a time, not all at once
affinity
reduce
negative cooperativity

Question 119

The T (taut) form is the conformation of the enzyme that has ______ affinity for the substrate and will therefore catalyze
______ (but not zero, as we see with the sequential model). The R (relaxed) form is the conformation in which the
enzyme has ______ affinity for its substrate and will bind it with ease to catalyze _______.

Answer 119

very low, few reactions
much higher, many reactions

Question 120

An enzyme is a ______ protein

Answer 120

catalytic

Question 121

An apoenzyme is the ______ form of a complete enzyme that just needs its ______ present so that it can become a
holoenzyme. For example, hexokinase in glycolysis is a fully formed protein, but it needs a _____ in order to
carry out its reaction.

Answer 121

inactive, cofactor
magnesium ion

Question 122

A zymogen is an ______ that requires a modification in ________ in order to be activated. From the slides, we are provided with the example of _______ that undergoes hydrolysis via _____ to yield its pi-form that is then further digested to give its active ______. This is a feature of many _______ enzymes – it is a bad idea to have them be active in the pancreas where they are synthesized; let them do their work in the small intestine where the food is!

Answer 122

inactive protein
primary structure

chymotrypsinogen, trypsin
alpha form

digestive

Question 123

If K is the dissociation constant, would KR or KT be expected to be higher? Why?

Answer 123

We expect KT to be higher since this is a dissociation constant; the T form of the enzyme has much lower affinity for
substrate, so it is less likely to bind it and will therefore be more favored in equilibrium.

Question 124

How would you expect the “L” value for an allosteric enzyme to change in the presence of an inhibitor?

Answer 124

Given that L is the ratio of T/R form, an inhibitor will shift the equilibrium to favor the T state and therefore increase L.
On the slides, you can see that the curve shifts to the right when L increases; this is MORE cooperativity.

Question 125

Consider an equilibrium between the T and R form of an allosteric enzyme. As the R form binds substrate to
form the enzyme-substrate complex, what happens to the equilibrium? How does this explain allostery for this
system?

Answer 125

This all comes down to LeChatlier’s principle (again). Recall that FREE enzymes have an equilibrium between the T/R
state. When a substrate binds to an R state enzyme and forms [ES], the concentration of free R state enzyme is effectively
decreased, so the system needs to shift towards the R state in order to replenish the free R state enzymes. This explains
allosteric effects as the binding of substrate is initially slow (less substrate = less binding of substrate to form [ES] and
shift the equilibrium) but once the substrate concentration increases to an appreciable amount, the equilibrium will begin
to quickly shift away from the T state.

Question 126

Hydrolase: Catalyze reactions in which bonds are cleaved by adding _____.
Lyases: Catalyze reactions in which groups (H2O, CO2, NH3, etc) are _____

Answer 126

water
removed to form a double bond or are added to a double bond

Question 127

Answer these questions about the chymotrypsin mechanism:
a. Which amino acid of the catalytic triad acts as a nucleophile to attack a carbon on the substrate?
b. What role does aspartate play in the mechanism?

Answer 127

a. Serine

b. Donates hydrogen bonds to histidine to provide stability as it shifts between acidic/basic forms.

Question 128

Chymotrypsin mechanism:
The bond cleavage always occurs at the ______ side of one of these three residues:
Explain how the active site orients the peptide such that the cleavage always occurs on this side.

Bonus: explain how the oxyanion is stabilized in the oxyanion hole.

Answer 128

C-terminus

Tyrosine, Phenylalanine, or Tryptophan.

Hydrophobic pocket

Ser-195 and Gly-193 provide hydrogen bonds (donated) to the oxyanion to stabilize it.

Question 129

d. There are two tetrahedral intermediates. Why are they called this?

Answer 129

They are formed from nucleophilic attacks that take place on the carbonyl carbon and force the formation of an oxyanion.
Because they are unstable, they do not last long and usually result in the cleavage of another carbon-oxygen bond to restore the carbonyl form.

Question 130

e. The acyl-enzyme intermediate is formed after the peptide bond cleavage. Why is it important that the reaction continues? i.e., why can’t it stop here now that it has cleaved the peptide?

Answer 130

In order to perform another reaction, the enzyme’s original state must be achieved; as of now, it is not capable of performing another reaction. The primary goal is restoring serine to its hydroxyl form instead of being involved in
an ester bond.

Question 131

f. What molecule must enter the active site to continue the reaction after the acyl-enzyme is formed?
i. Does this molecule act as an acid or a base?
ii. In what other way does this molecule react?

Answer 131

i. Water must enter the active site. It will act as an acid and donate a proton to histidine to form a hydroxide ion.
ii. The hydroxide ion that is formed will be a strong nucleophile that attacks the carbonyl carbon on the acyl-enzyme molecule and form another tetrahedral intermediate that breaks down to release the serine and restore the enzyme to its
original state