Enzymes Lecture 3

Question 1

Note: For competitive inhibitions Km stays the same because α = α’ (equation for apparent Km is = α*Km/α’)

Answer 1

!

Question 2

How can we learn more about the way an enzyme

binds a substrate molecule in a reaction?

Answer 2

We can study the inhibitory effect of small molecules (other than the substrate) on the reaction.

Question 3

______ are important pharmaceutical agents

For example, _____ inhibits the enzyme that catalyzes the first step in the synthesis of prostaglandins (involved in some processes that produce pain)

Answer 3

Enzyme Inhibitors

Aspirin

Question 4

There are two main classes of inhibitors.

This one is noncovalent binding of the inhibitor; can be reversed by removing the inhibitor

What is one common type?

Answer 4

Reversible Inhibition

Competitive

Question 5

There are two main classes of inhibitors.

These bind covalently with or destroy an essential functional group on an
enzyme, or form a very stable noncovalent
association

Answer 5

Irreversible Inhibition

Question 6

This type of inhibitor molecules competes with the substrate molecule for the SAME active site of an enzyme

The inhibitor is very similar to the

Answer 6

Competitive Inhibitor

Substrate

Question 7

By binding to the enzyme’s active site, the
inhibitor prevents the substrate from binding.

It forms an _____ complex and increases/decreases the rate of formation of ES complex.

Can the inhibitor undergo catalysis?

Answer 7

EI complex

decreases the rate of formation of ES complex

No.

Question 8

Competitive inhibitors can be analyzed by _____ kinetics

The M-M equation becomes…

At high [S], the ES complex is UNFAVORED/FAVORED so
that the maximum rate can be obtained?

Answer 8

Steady State Kinetics

Vα = Vmax[S]/(αKm + [S])

Favored

Question 9

What is the equation for α?

What is αKm?

Answer 9

α = 1 + [I]/KI and KI =[E][I]/[EI]

The apparent Km

Question 10

Figure 11.2 describes the effects of competitive inhibition on enzyme kinetics

____ is the same at high [S]

Lineweaver-Burke plots are _____ as expected for steady state kinetics

Answer 10

Vmax

Linear

Question 11

Is Vmax changed by the presence of a competitive inhibitor?

Answer 11

No, it stays the same (the y-intercept is the same)

Question 12

An example of a competitive inhibitor is
shown in Fig 11.3. The molecule UpA is a very good substrate for the enzyme ribonuclease

If the oxygen atom at the cleavage site in the
substrate UpA is replaced by a ____ group to form a phosphonate analog UpcA, a strong competitive inhibitor is formed.

Ribonuclease binds the analog, but it cannot
cleave the _____ bond.

Answer 12

CH2

phosphonate bond

Question 13

Another example of a competitive inhibitor, this is used in medicine as a treatment for patients who have ingested methanol

It competes effectively with methanol as an alternative substrate for ________

Answer 13

Ethanol

alcohol dehydrogenase

Question 14

This is another type of reversible inhibitor, it is seen only with enzymes having two or more substrates

It binds at a site different from the substrate active site

Answer 14

Uncompetitive Inhibitor

Question 15

Unlike a competitive inhibitor, an uncompetitive inhibitor binds only to the ____ complex forming an

Answer 15

Uncompetitive

ES complex

ESI complex

Question 16

In the M-M equation, in the presence of an uncompetitive inhibitor, the equation is altered to….

Where α’ =

Answer 16

Vα= Vmax[S]/(Km + α’[S])

α’ = 1 + [I]/KI’ and KI’=[ES][I]/[ESI]

Question 17

Does an uncompetitive inhibitor lower or increase Vmax?

Does the apparent Km increase or decrease? Why?

Answer 17

Lowers Vmax

Decreases because the [S] needed to reach 0.5 Vmax increases by the factor α’

Question 18

This type of inhibitor binds at a site different from that of the substrate active site, but it binds to either E or ES

Answer 18

Mixed Inhibitor

Question 19

What is the rate equation describing mixed inhibition?

Answer 19

Vo = Vmax[S]/αKm + α’[S]

Question 20

How does mixed inhibition affect Km and Vmax?

Answer 20

A decrease in the apparent affinity of the enzyme for the substrate (a decrease in apparent affinity means the Km value appears to increase) or or in an increase in the apparent affinity (an increase in apparent affinity means the Km value appears to decrease) when the inhibitor binds favorably to the enzyme-substrate complex. In either case the inhibition decreases the apparent maximum enzyme reaction rate

Question 21

When α=α’, it is known as ______ inhibition.

Note this is a special case of mixed inhibition

Answer 21

Noncompetitive

Question 22

What is the only thing affected during noncompetitive inhibition?

Answer 22

Vmax

Question 23

What equation can be used to summarize the effects of all reversible inhibitors?

Answer 23

Vo = Vmax[S]/αKm + α’[S]

Question 24

This category of inhibitors binds covalently with enzymes or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or form a very stable noncovalent association

Answer 24

Irreversible Inhibitors

Question 25

In one example of irreversible inhibition, ___ reacts with diisopropylfluorophosphate (DIFP) and irreversibly inhibits the enzyme

This has led to the finding that ______ is the key acctive site Ser residue in the above molecule

Answer 25

Chymotrypsin

Ser195

Question 26

These are a special type of irreversible inhibitor that are unreactive until they bind to the active site of a specific enzyme

Answer 26

Suicide Inactivator

Question 27

The suicide inactivator undergoes the first
few steps of the reaction, but then it’s
converted into a reactive compound that
combines irreversibly with the enzyme.

They are important in drug design

Answer 27

!

Question 28

Enzymes have an optimal ____ for enzyme activity

Above or below the optimal value, activity decreases

Answer 28

pH

Question 29

An ionic interaction essential for stabilization of the enzyme’s active conformation might be eliminated.

For example, consider removing a proton from a his residue. The pH range over which an enzyme undergoes changes in activity can provide information as to the type
of amino acid involved

Answer 29

!

Question 30

____ is a digestive enzyme located in the small intestine

Answer 30

Chymotrypsin

Question 31

Chymotrypsin catalyzes the hydrolytic cleavage of ____ bonds

Answer 31

Peptide

Question 32

Chymotrypsin is specific for peptide bonds adjacent to _____ residues

Answer 32

Aromatic AA resiudes (Trp, Phe, Tyr)

Question 33

Chymotrypsin has a widely studied mechanism which includes….

Answer 33

1) General acid-base catalysis
2) Covalent catalysis
3) Transition-state stabilization

Question 34

Chymotrypsin does not catalyze a direct attack
of water on the peptide bond; a transient
covalent ______ intermediate is formed

Answer 34

acyl enzyme

Question 35

What are the two phases of the chymotrypsin mechanism?

Answer 35

Acylation, deacylation.

Question 36

This phase of the chymostrypsin mechanism cleaves peptide bond and form an ester linkage between peptide carbonyl carbon and the enzyme

Answer 36

Acylation

Question 37

This phase of the chymostrypsin mechanism hydrolyzes the ester linkage and regenerate the nonacylated enzyme

Answer 37

Deacylation