203 Classic Enzyme Mechanisms

Question 1

General acid-base catalysis involves…

Answer 1

a proton transfer between a weak acid or a weak base other than water

an active-site residue can donate a proton I

Question 2

An active-site residue that can donate a proton is classified as?

Answer 2

a general acid and the catalysis is general acid catalysis

Question 3

An active-site residue that accepts a proton is classified as what?

Answer 3

a general base and this is a general base catalysis

Question 4

TRUE OR FALSE: reactions can only be either acid or base catalysis?

Answer 4

FALSE: Certain reactions may be subjected simultaneously to both processes, that is acid catalysis and base catalysis, and have concerted general acid‐base catalysis.

ex: amino acids with side chains that have acidic or basic groups serve as good proton donors or acceptors.

Question 5

Is histidine a good proton donor or acceptor at physiological pH?

Answer 5

Histidine is special in that it can behave either as a donor or acceptor at physiological pH.

Question 6

What is covalent catalysis?

Answer 6

in covalent catalysis, there is a transient state in which substrate and enzyme are covalently bonded. This type of catalysis involves both nucleophilic and electrophilic stages

Question 7

a good covalent catalyst should have high nucleophilicity but at the same time have the ability to be a good leaving group. Why is that?

Answer 7

You want the transient state to be stable, via formation of the covalent bond, but it should be able to break down to form the final product and free enzyme. Groups with highly mobile electrons such as imidazole and thiol groups have these properties; therefore His and Cys make good covalent catalysts.

Question 8

in metal catalysis, metal ions help…

Answer 8

orient the substrate properly, mediate oxidation-reduction reactions, shield negative charges (=electrostatically stabilize the reaction)

Question 9

What occurs in electrostatic catalysis?

Answer 9

the transition state is stabilized through non‐covalent bond interactions between enzyme and substrate. By excluding water from the active site of the enzyme, the dielectric constant is lowered, and in such an environment, the electrostatic interactions are much stronger than in an aqueous solution. The charge distribution not only stabilizes the transition state but may also serve to guide the substrate to the binding site, which can help explain how certain enzymatic reactions have rates that are greater than the diffusion‐controlled limits.

Question 10

What is the proximity and orientation effect?

Answer 10

this means the spatial arrangement of substrate and enzyme must be proper for the reaction to occur. Although proximity alone may contribute very little to catalysis, proper orientation and immobilizing the transition state can result in significant rate enhancements. Enzymes binding to substrates not only aligns them but also immobilizes them, thereby optimizing reactivity.

Question 11

Why does the enzyme bind to the transition state w/much higher affinity than either to the substrate or product?

Answer 11

By doing so, the concentration of the transition state increases, thereby increasing the rate of the reaction. Transition state analogs, which are stable molecules that resemble the actual transition state, serve as potent competitive inhibitors of the enzyme.

Question 12

What are hydrolases?

Answer 12

enzymes that catalyze hydrolysis.

Question 13

What are the biological functions of proteases aka peptidases?

Answer 13

• digestion (chymotrypsin, trypsin, elastase, pepsin)
• blood clotting
• immune system
• apoptosis
• protein turn-over

Question 14

All proteases do the same thing: what is this?

Answer 14

they catalyze the hydrolysis of the peptide bond

-this is done to the scissile peptide bond - this bond is cleaved between the carbonyl of the N-terminal product peptide and the amino group of the C-terminal peptide product

Question 15

What are the two classes of proteases? what is classification based on?

Answer 15

• endoproteases
• exoproteases
• classification of different endoproteases is based on the type of residue that is key to the catalytic mechanisms

Question 16

endoproteases do what?

Answer 16

cleave internal peptide bonds

Question 17

exoproteases do what?

Answer 17

cleave peptide bonds of the terminal amino acids

Question 18

Serine proteases are important in what?

Answer 18

digestion and contain an active site Ser and Bacillus subtilis enzyme subtilisin

Question 19

How are chymotrypsin and subtilisin similar and different?

Answer 19

SIMILAR: the catalytic triad at the active site has structure is similar to chymotrypsin

DIFFERENT the primary and tertiary structure of subtilisin is different

Question 20

What are the types of endoproteases discussed in this lecture?

Answer 20

Serine (chymotrypsin, trypsin, elastase)

cysteine

aspartyl

metalloproteases: Zn2+ dependent

Question 21

What are the family members in Cys proteases?

Answer 21

papain (found in papaya and caspases (named for the cys active site residue and b/c they only cleave after an Asp residue )

Question 22

Which proteases are Asp-dependent?

Answer 22

cathepsins and the HIV protease

Question 23

What are zymogen/proenzyme?

Answer 23

they are digestive enzymes that are made in the pancreas and are in their inactive form.

-this is necessary b/c otherwise they would destroy the cells that make them.

Question 24

How are pancreatic zymogens activated?

Answer 24

only after they reach the small intestine, where they can digest proteins in the food we have consumed. Zymogens are activated by some biochemical process, often via proteolysis. The cleavage causes a change in the conformation so that the active site is exposed.

Question 25

Is the activation of zymogens/proenzymes reversible or irreversible?

Answer 25

irreversible, hence other methods are used to inhibit these proteases, such as inhibitor proteins (e.g., the pancreatic trypsin inhibitor). these bind w/very high affinity to the enzyme active site thereby inactivating the enzyme

Question 26

What is trypsin? How is it related to chymotrypsin?

Answer 26

an enteropeptidase (entero = intestinal) secreted by the intestinal cells, cleaves a short hexapeptide from trypsinogen to form trypsin, which in turn activates the precursors of chymotrypsin, carboxypeptidases, elastase as well as trypsinogen itself.

Question 27

How is the disease acute pancreatitis caused?

Answer 27

when the normal pathway by which the pancreatic secretions reach the intestines are blocked. As a result zymogens get activated in the pancreatic cells themselves and the active proteases then attack the pancreatic tissue. This causes severe pain and can be fatal

Question 28

What happens when the polypeptide that is to be cleaved binds to the protease?

Answer 28

the residue that is on the N-terminal side of the scissile bond fits into the substrate-binding pocket that is w/in the active site. the serine protease then cleaves the amide bond that is on the C-terminal side of this residue

Question 29

How is specificity of a protease defined?

Answer 29

each protease has a specificity pocket that accommodates certain residues - this defines specificity

Question 30

In the case of trypsin, how is the specificity pocket?

Answer 30

In the case of trypsin, the Asp at the bottom of the pocket provides binding to positively charged side chains. Hence trypsin cleaves after lysine and arginine (but not histidine, b/c it does not fit in the pocket).

Question 31

How is the binding pocket for chymotrypsin?

Answer 31

the binding pocket for chymotrypsin is able to hold bulkier non-polar, hydrophobic side chains such as Phe, Tyr, Trp and Met (but rarely met)

Question 32

How is the binding pocket for Elastase?

Answer 32

elastase has a narrow shallow specificity pocket and the enzyme elastase cuts after Gly, Ala, and Val and these are the residues that can fit.

Question 33

What differentiates between the enzymes trypsin, chymotrypsin, and elastase specificity?

Answer 33

their pocket specificity - and this also serves to position the active site serine close to the carbonyl group of the scissile bond

Question 34

What is the catalytic triad?

Answer 34

Phe, Ser, and Asp In the first step of binding Phe is in the selectivity hydrophobic pocket Ser is poised to act as a nucleophile b/ the adjacent His is removing the Ser proton The resulting positive charge on the His is stabilized by the adjacent negatively-charged side chain, the third player in the catalytic triad. there is an ionic bond between the His N-H and the carboxylate group on Asp 102

Question 35

How does ser 195 act as a nucleophile?

Answer 35

it attacks the carbonyl group of the scissile bond to produce a tetrahedral acyl intermediate as well as a negative charge on the carbonyl carbon of the substrate.

Question 36

After ser 195 acts as a nucleophile attacking the carbonyl group and producing a negative charge on the carbonyl carbon substrate, how is the negative charge stablized?

Answer 36

by the H-bonding w/the glycine 193 in the oxyanion hole

Question 37

When breaking the scissle bond, why is the tetrahedral intermediate a short-lived intermediate?

Answer 37

b/c the negative charge on the carbonyl oxygen is unstable and will result in collapse of the intermediate. A double bond is formed between this oxygen and the carbon, displacing the bond between the carbon and the amino group of the peptide bond. This breaks the peptide bond. The amino group on the leaving peptide is protonated by the His 57, thus helping its release as a free peptide.

Question 38

After the tetrahedral intermediate collapses during the sciccle bond breakage, what occurs?

Answer 38

The acyl enzyme intermediate that is formed when the intermediate collapses and the petide bond is cleaved is then attacked by the strongly nucleophilic OH portion of water. This generates a second tetrahedral intermediate with the same oxyanion hole stabilizing its negative charge.

Question 39

Why is the second tetrahedral intermediate in the scissle breakage short lived?

Answer 39

b/c it undegoes deacylation to form a carboxylate anion. The Ser 195 accepts a proton from the adjacent imidazole group of His 57 as the second product dissociates from the enzyme. The enzyme is now in its original form ready to accept another substrate molecule of proteolytic cleavage

Question 40

What are lysozymes?

Answer 40

lysozymes act as antibacterial enzymes b/c they break down the peptidoglycan layer that is found in the cell wall of bacteria.

Question 41

In what biological fluids can you find lysozyme?

Answer 41

fluids ranging from tears to egg whites as well as phage-escape mechanisms.

Question 42

What are peptidoglycans/

Answer 42

polymers made up of N-acetyl glucosamine and N-acetyl muraminic acid

Question 43

What is Chitin?

Answer 43

another substrate of lysozyme, is a NAG polymer and is found in insects such as beetles and cockroaches as well as in crustaceans such as lobsters and crab

Question 44

what is an example of convergent evolution?

Answer 44

the fact that lysozymes are found in different species, many of which are unrelated e.g. bacteriophage T4 and humans, and yet carry out similar functions

Question 45

Hen egg white lysozyme (HEWL) was the first enzyme to have what?

Answer 45

its crystal structure solved

Question 46

what does the crystal structure of hen egg white lysozyme (HEWL) tell us?

Answer 46

the active site of HEWL is a deep crevice 6 residues of the polymer bind at the active site A, B, C, D, E, F