Willmott 6 enzymes

Question 1

Active site:

Answer 1

small region where reaction occur. Microenvironment e.g. excludes water.

Question 2

Large Kcat=

Answer 2

good turnover

Question 3

Small Km=

Answer 3

only needs small amount of substrate/high affinity

Question 4

Ratio of Kcat/Km:

Answer 4

convenient measure of enzyme efficiency

Question 5

Specificity:

Answer 5

Complementarity: enzyme & substrate

Question 6

Irreversible inhibitors:

Answer 6

bind v. tight, covalent bonds e.g. nerve gas

Question 7

Reversible inhibitors:

Answer 7

non-covalent

Question 8

Competitive inhibitors:

Answer 8

bind active site, Vmax unaffected, approaches same Vmax if sub conc high enough, Km increased

Question 9

Non-Competitive inhibitors:

Answer 9

bind another site. Vmax decreased, inc sub conc does not displace inhibitors Km unaffected

Question 10

Ribonuclease:
No. AA;s
Function:

Answer 10

• 124 amino acids (13.7 kDa)
• Hydrolyses phosphodiester bonds in RNA
• Releases free 3’-phosphate and 5’-hydroxyl termini
• Bell-shaped pH curve with optimum ~pH
• Catalysis by pair of histidine residues 12 & 119

Question 11

Base-catalysed hydrolysis of RNA

Answer 11

1) nucleophillic attack
2) intermediate (5bonds) – unstable
3) phosphate & base
In absence of enzyme = alkaline & times

Question 12

Mechanism of ribonuclease

Answer 12

HAs= H+

NO= H+
Lys structural role/ not catalytic
1 His 12 attack & take H+ => O- => P
2 stablises intermediate by lys

Question 13

Evidence for mechanism of ribonuclease:

x3

Answer 13

-pH dependence of reaction => His pKa of 6.5
- use of small molecule inhibitor e.g. iodoacetate covalently modify
Phosphate can’t be processed by enzymes so sites in active site
- crystaliseation shows key His’s
-site-direct mutagenesis -> convert => alkaline
Use of non-hydrolysable substrate analog e.g. phosphate ester can be processed