Enzymes Lecture 4

Question 1

This is a digestive enzyme lpsinocated in the small intestine

Answer 1

Chymotrypsin

Question 2

Chymotrypsin catalyzes the hydrolytic of ______ bonds in proteins (a protease)

It is specific for peptide bonds adjacent to ______ AA residues

Answer 2

Peptide bonds

Aromatic (trp, phe, tyr)

Question 3

What are the 3 steps of the chymotrypsin mechanism

Answer 3

General acid-base catalysis
• Covalent catalysis
• Transition-state stabilization

Question 4

Does chymotrypsin catalyze a direct attack of water on the peptide bond?

What occurs?

Answer 4

No.

A transient covalent acyl enzyme intermediate is formed

Question 5

The chymotrypsin reaction has two phases

Answer 5

Acylation and Deacylation

Question 6

This step of the chymotrypsin mechanism cleaves the peptide bond and forms an ester linkage between peptide carbonyl carbon and the enzyme

Answer 6

Acylation

Question 7

This phase of the chymotrypsin hydrolyzes the ester linkage and regenerates the nonacylated enzyme

Answer 7

Deacylation

Question 8

What is the primary structure of chymotrypsin?

Answer 8

3 polypeptide chains linked by several disulfide bonds

Question 9

Which three AAs are important for catalysis?

How do they occur in 3D structure?

Answer 9

His57, Asp102, Ser195

Grouped together

Question 10

Evidence for a covalent acyl enzyme intermediate came from ______ kinetics

In this experiment, a small ester (p-nitrophenylacetate) was hydrolyzed by chymotrypsin.

Hydrolysis was determined by measuring the release of ____

The reaction proceeded with a rapid initial phase, which is which phase?

Then it was followed by a slower phase, which was which phase?

Answer 10

Pre-steady state kinetics

p-nitrophenol

Acylation

Deacylation

Question 11

In terms of the effect of pH on chymotrypsin catalyzed reactions, a maximum rate is observed at about pH =

In fig 6-20a, the rate plotted is at low (subsaturing) [S] so it represents

Decreased Kcat at low pH results from protonation of _____

Where does the proton come from? (acid-base catalysis)

Answer 11

8

Kcat/Km

His57

Ser

Question 12

A plot of 1/Km also shows pH dependence of chymotrypsin, there is a decline after pH =

The α-amino group of _____ loses a proton at a high pH

It is the __ terminal end of one of chymotrypsin’s chains

Answer 12

8

Ile16

Amino terminal

Question 13

Ser, His, and asp are linked in a hydrogen bonding network called a

Answer 13

Catalytic triad

Question 14

In acylation (at the beginning) the substrate (S) is a polypeptide containing an aromatic amino acid ADJACENT to the ___ bond to be cleaved

The polypeptide binds to the _______ of chymotrypsin

The aromatic amino acid residue next to the peptide bond to be cleaved sits in a ___ pockey on the enzyme

Answer 14

Peptide bond

Active site

Hydrophobic

Question 15

What is the nuclephile in the acylation phase?

Answer 15

Ser195

Question 16

When a peptide substrate binds to chymotrypsin, the hydrogen bond between ___ and ____ is compressed, the pKa of his increases from 7 to 12

Acting as a general base, ____ can remove the proton from the ser OH

Answer 16

Asp and His

His

Question 17

In the acylation phase, interaction of ser and his creates a strong nucleophilic alkoxide ion on ____

The positively charged __ group of his is stabilized by the negatively charged ___ group of asp

Answer 17

Ser 195

Carboxylate

Question 18

In the second step of the acylation phase, formation of a ____ ______ state occurs

The alkoxide ion from Ser 195 attacks the peptide ___ group forming this short lived acyl-enzyme intermediate

Answer 18

Tetrahedral transition state

Carbony

Question 19

Note in the mechanism figure that a short-lived
negative charge on the carbonyl oxygen of the
substrate is generated. This charge is stabilized by H bonds from the amide group of gly 193 and one other residue.

Answer 19

!

Question 20

Note - the H bond from Gly 193 is present ONLY in the _____ and in the _____ states for its formation and breakdown

What it do for these states?

Answer 20

Intermediate and transition

Reduces the energy required to reach them

Question 21

Gly 193 reducing the energy required to reach intermediate and transition states is an example of ________ energy in catalysis

Answer 21

Binding energy

Question 22

In step 3 of the acylation mechanism, what occurs?

Reformation of a ____ bond between the carbonyl oxgen and carbon displaces the bond between the carbon and amino group of the peptide linkage, breaking the ______

The amino group of the peptide bond is protonated by _____ and diffuses away from the enzyme’s active site, leaving an acyl-enzyme intermediate with it’s aromatic AA residue in the hydrophobic pocket of the enzyme

Answer 22

Formation of the acyl -enzyme intermediate

His 57

Question 23

In step 4 (deacylation phase), an incoming water molecule is deprotonated by general ___ catalysis

The imidazole group of _____ accepts a proton from

This results in a ___ nucleophile

Answer 23

Base

His57, Water

Hydroxide

Question 24

In step 5 (deacylation phase), what occurs?

The ___ ion attacks the ___ linkage of the acyl enzyme

This generates a short lived tetrahedral intermediate

What once again becomes negatively charged?

Answer 24

Formation of a second tetrahedral transition state

Hydroxide attacks the ester linkage

The oxygen

Question 25

In step 6, the tetrahedral intermediate formed in step 5 collapses and forms a ____ ______

What is displaced?

Answer 25

Carboxylate anion

Ser 195

Question 26

In step 7, diffusion of the second product from the active site of the enzyme regenerates the free enzyme.

Answer 26

!

Question 27

What does the triad of AAs and the H-bonding groups do for the chymotrypsin reaction?

How?

Answer 27

Lowers the activation energy

Stabilizing transition states

Question 28

What group of cysteine in thiol proteases like papain functions in a similar way to the hydroxyl group of serine?

The binding of a metal ion to the imidazole of his 57 or the thiolate of cys in papain will do what?

Answer 28

Thiol (SH) group

Inhibit the reaction

Question 29

These are common non-competitive inhibitors of many enzymes

These metal ions form strong complexes with
neutral and anionic groups of amino acid side
chains which are important to enzyme
catalysis (such as the imidazole of his or the
thiolate of cys)

Answer 29

Heavy metal ions (Pb2+, Ag+, Hg+)

Question 30

________ ,a bisubstrate enzyme, catalyzes the reversible reaction of β-D-glucose to glucose-6-phosphate with Mg·ATP converted to Mg·ADP

Answer 30

Yeast hexokinase

Question 31

ATP and ADP always bind to enzymes as a complex with the metal ion ______

The γ-phosphoryl of ATP is transferred to the
OH at C-6 of glucose.

Water freely enters the enzyme active site,
but hexokinase favors reacting with the OH of
glucose over the OH of water by a factor of
106.

Answer 31

Mg2+

!

Question 32

Hexokinase disciminates between water and glucose because of a ______ in the enzyme when the correct substrate binds

This is a good example of _____

When glucose is absent, hexokinase is in an inactive conformation

Answer 32

Conformational Change

Induced fit

Question 33

When glucose and Mg·ATP bind, the binding

energy induces a conformational change to the catalytically active form of _____

Answer 33

hexokinase

Question 34

This is a glycolytic enzyme

It catalyzes the reversible _____ of 2-phosphoglycerate to phosphoenolpyruvate

What type of catalysis is this?

How many steps?

Then enolic intermediate is stabilized by what?

Answer 34

Enolase

Dehydration

Metal ion catalysis

Two steps

Two Mg2+ ions

Question 35

A natural bacterial agent found in tears and egg whites

Answer 35

Lysozyme

Question 36

This enzyme cleaves the B1–>4 glycosidic C-O bond between two different sugar residues the the molecule N acetylmuramic acid and N acetylglucosamine.

What type of reacion is it?

Which type is supported?

Answer 36

Lysozyme

Nucleophilic substitution

SN2