203 Classic Enzyme Mechanisms Flashcards Preview

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Flashcards in 203 Classic Enzyme Mechanisms Deck (46)
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1
Q

General acid-base catalysis involves…

A

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

an active-site residue can donate a proton I

2
Q

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

A

a general acid and the catalysis is general acid catalysis

3
Q

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

A

a general base and this is a general base catalysis

4
Q

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

A

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.

5
Q

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

A

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

6
Q

What is covalent catalysis?

A

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

7
Q

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?

A

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.

8
Q

in metal catalysis, metal ions help…

A

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

9
Q

What occurs in electrostatic catalysis?

A

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.

10
Q

What is the proximity and orientation effect?

A

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.

11
Q

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

A

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.

12
Q

What are hydrolases?

A

enzymes that catalyze hydrolysis.

13
Q

What are the biological functions of proteases aka peptidases?

A
  • digestion (chymotrypsin, trypsin, elastase, pepsin)
  • blood clotting
  • immune system
  • apoptosis
  • protein turn-over
14
Q

All proteases do the same thing: what is this?

A

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

15
Q

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

A
  • endoproteases
  • exoproteases
  • classification of different endoproteases is based on the type of residue that is key to the catalytic mechanisms
16
Q

endoproteases do what?

A

cleave internal peptide bonds

17
Q

exoproteases do what?

A

cleave peptide bonds of the terminal amino acids

18
Q

Serine proteases are important in what?

A

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

19
Q

How are chymotrypsin and subtilisin similar and different?

A

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

DIFFERENT the primary and tertiary structure of subtilisin is different

20
Q

What are the types of endoproteases discussed in this lecture?

A

Serine (chymotrypsin, trypsin, elastase)

cysteine

aspartyl

metalloproteases: Zn2+ dependent

21
Q

What are the family members in Cys proteases?

A

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

22
Q

Which proteases are Asp-dependent?

A

cathepsins and the HIV protease

23
Q

What are zymogen/proenzyme?

A

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.

24
Q

How are pancreatic zymogens activated?

A

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.

25
Q

Is the activation of zymogens/proenzymes reversible or irreversible?

A

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

26
Q

What is trypsin? How is it related to chymotrypsin?

A

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.

27
Q

How is the disease acute pancreatitis caused?

A

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

28
Q

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

A

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

29
Q

How is specificity of a protease defined?

A

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

30
Q

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

A

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).

31
Q

How is the binding pocket for chymotrypsin?

A

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)

32
Q

How is the binding pocket for Elastase?

A

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.

33
Q

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

A

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

34
Q

What is the catalytic triad?

A

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

35
Q

How does ser 195 act as a nucleophile?

A

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.

36
Q

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?

A

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

37
Q

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

A

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.

38
Q

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

A

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.

39
Q

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

A

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

40
Q

What are lysozymes?

A

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

41
Q

In what biological fluids can you find lysozyme?

A

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

42
Q

What are peptidoglycans/

A

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

43
Q

What is Chitin?

A

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

44
Q

what is an example of convergent evolution?

A

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

45
Q

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

A

its crystal structure solved

46
Q

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

A

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