RNase A/Lysozyme (Proff. Rice)

Question 1

What is the function of RNase A?

Answer 1

It cleaves ssRNAs

Question 2

In which organ is it found in abundance?

Answer 2

The pancreas

Question 3

Which bond does it cleave?

Answer 3

The P-O5’ bond

Question 4

RNase A is specific to cut after which bases?

Answer 4

Pyrimidines (U or C)

Question 5

The reaction was discovered to be a two step reaction. The first step is the formation of which intermediate?

Answer 5

2’-3’-cyclic nucleotide

Question 6

pH studies showed that the reaction is greatly pH dependant due to which critical residue?

Answer 6

Histidine

Question 7

How many critical Histidine residues are there?

Answer 7

Two

Question 8

What do the two His residues act as?

Answer 8

One as a general acid, the other as a general base

Question 9

Iodoacetate treatment typically modifies which residue?

Answer 9

Cys

Question 10

In RNase A, iodoacetate leads to activity loss. It was wrongly concluded this was due to Cys modification. Which residue was actually effected?

Answer 10

Highly reactive His residues.

Question 11

What is the tertiary structure?

Answer 11

A 3-stranded V-shaped anti-parallel B-sheet.

Question 12

What can the active site be described as?

Answer 12

A deep cleft

Question 13

The specificity pocket was determined using a crystal structure of a complex and a pyrimidine nucleotide product. What is the product?

Answer 13

Cytidine-3’ Phosphatte

Question 14

In the active site, what do His12 and His119 do?

Answer 14

They’re directly involved in the reaction mechanism.

Question 15

In the active site, what does Lys41 do?

Answer 15

Stabilises the -ve phosphate in the intermediate.

Question 16

Other basic residues (Lys, Arg) assist what?

Answer 16

RNA binding

Question 17

In the pocket, what does Phe120 do?

Answer 17

Forms vdW contacts with the RNA base

Question 18

In the pocket, what do Ser123 and Thr45 do?

Answer 18

H bond

Question 19

Why can’t purines fit in the pocket?

Answer 19

They’re too large

Question 20

Which residue in the pocket can act both as an H bond acceptor/donor?

Answer 20

Thr45

Question 21

What is Angiogenin?

Answer 21

A homologue of RNase A

Question 22

What does Angiogenin do?

Answer 22

Promotes development of blood vessels in healthy tiessues + tumours

Question 23

What does Lysozyme do?

Answer 23

It cleaves peptidoglycan.

Question 24

What is peptidoglycan?

Answer 24

A polypeptide-polysaccharide complex found in gram+ve bacterial cell walls.

Question 25

Where is lysozyme found?

Answer 25

In most human bodily secretions (inc. tears and nasal mucus).

Question 26

What is the structure of peptidoglycan?

Answer 26

(NAG-NAM)n polymer with cross links between NAMs.

Question 27

What is special about the peptidoglycan cross links?

Answer 27

They contain the unusual amino acids D-ala and D-Glu.

Side chain D-Glu is bonded to the main chain of Lys.

The side chain of Lys ib bonded to with main chain of Gly.

Question 28

What enzymes form the cross links?

Answer 28

D-Ala-D-Ala transpeptidases. (The targets of penicillin/related antibiotics)

Question 29

Where does lysozyme cleave peptidoglcan, and why?

Answer 29

Between NAM and NAG in regions where there are not too many cross links. This is because the cross links sterically interfere with the enzyme.

Question 30

What is the basic structure of lysozyme?

Answer 30

129AAs, 4SS bridges.

Two domains separated by a deep cleft:

a small hydrophobic ß-sheet domain

a hydrophobic core surrounded by short α-helices

Question 31

What was observed binding to the top half of the lysozyme cleft?

Answer 31

triNAG

Question 32

Below is a table of the rate of hydrolysis of poly NAG substrates by lysozyme. What can be concluded from the data about the active site?

Substrate | Rate

(NAG)2 0

(NAG)3 1

(NAG)4 8

(NAG)5 4000

(NAG)6 32000

(NAG)8 32000

Answer 32

The active site binds six NAG sugars, as there was no increase from (NAG)6 to (NAG)8

Question 33

How many sugars can lysozyme bind, and where does it cleave them?

Answer 33

6 sugars. Clevage occurs after the 4^th, giving A-B-C-D and E-F as products.

Question 34

What are the two catalytic residues found in the lysozyme active site?

Answer 34

Glu35, Asp52

Question 35

Both Glu and Asp have carboxylic acid groups. In lysozyme, what states to Glu35 and Asp52 exist in?

Answer 35

Glu35: Protonated as it is in a hydrophobic microenviornment.

Asp52: normal (-ve resonance). [pH ~6, Asp pKa=3.5]

Question 36

Describes the lysozyme catalyses mechanism.

Answer 36

1. Asp52 nucleophilically attacks the NAM C₁ forming a covalent glycosyl-enzyme intermediate.
2. Glu35 protanates the (NAG-NAM) (E-F) which diffuses away.
3. Water attacks the NAM (D) C₁, giving it an OH, whilst its bond to Asp52 breaks. The remaing H⁺ is donated to Glue35 (reprotonation).

Question 37

Other glycosidases have evolved, yet share no sequence similarity to lysozyme. What do they share?

Answer 37

Catalytic carboxyl groups in the same relative positions.

Question 38

Give examples of enzymes that break down long sugars into smaller units.

Answer 38

• Lysozymes - break down bactieral cell walls
• Lactases - break down lactose
• Amylases - break down starch
• Cellulases - break down cellulose to glucose
• Neuraminidases - penetrate cell walls (viruses/bacteria)