BMP: Enzyme specificity

Question 1

What is the active site?

Answer 1

The three dimentional catalystic site at which enzyme-substrate interaction occurs

Question 2

What 2 models are used to describe enzyme-substrate interaction?

Answer 2

1. Lock and key - The enzyme and substrate are shaped to fit perfectly
2. Induced fit - active site and substrate alter shape to connect

Question 3

Compare the structures of mannose. glucose and fructose

Answer 3

Question 4

Why is sodium fluoride added to the blood sample?

Answer 4

TO prevent glycolysis - prevent break down of glucose

Question 5

What is normal resting blood glucose levels?

Answer 5

70-100 mg/dl

Question 6

What is an example of a condition where blood glucose levels differ?

Answer 6

Diabetes melitus

Question 7

What is glycated haemoglobin (HbA1c) and what is the desirable range for healthy adults?

Answer 7

when haemoglobin joins to glucose in the blood

below 42mmol/L

Question 8

What is substrate specificty determined by?

Answer 8

• Structure
• Charge
• Polarity
• hydrophobicity

of the substrate binding molecule

Question 9

Give an example of a highly specific enzyme and unspecific enzyme and its substrates

Answer 9

Specific: Ureases - urea

unspecific: serine proteases

Question 10

What is the enzyme used in this experiement to demonstrate enzyme specificity

Answer 10

Glucose oxygenase

Question 11

1. What type of protein is glucose oxygenase?
2. What is its molecular weight?
3. What cofactor is attached?

Answer 11

1. Dimeric protein
2. 160kDa
3. flavin adenine dinucleotide to each monomer

Question 12

Describe the oxidation of glucose

Answer 12

1. B-D-Glucose + O2 —(glucose oxygenase)–> 5-lactone + H2O2 + B-D-glucono-1
2. 5-Lactone + H2O2 —(H20)–> D-gluconic acid + H2O2
3. H2O2 + AH2 (dye) —(Peroxidase)–> A (coloured compound) + 2H2O

The coloured compound on oxidation can be measured spectrophotomerically

The dye used is ABTS and the intesnity of colour is directly proportional to glucose concentration in the blood - measure glucometer or spectrophotomerically

Question 13

What is the enzyme solution made off?

Answer 13

phosphate buffer (pH7), Peroxidase, Glucose oxidase (GOD), ABTS

Question 14

What is the blank made off and whta wavelength is it zeroed at?

Answer 14

• 0.2ml water and 5ml enzyme solution
• 436nm

Question 15

Describe the procedure to determine enzyme specificty

Answer 15

1. Substrate solution (0.005M of M,F,G) and enzyme solution added together. Transfer sample directly to spectophotomoeter for zero reading.
2. Repeat every minute for 10 minutes.
3. Repeat for each substrate and ensure blank redone each time
4. PLot graph of absorbance against time for 3 substrates
5. Calculate the gradient for each substrate (linear section) and determine 7oxidation (assume O2 100%)

Question 16

Rate oxidation of the sugars from highest to lowest. What can be said about enzyme spcificity?

Answer 16

G, M, F

Glucose oxygenase have greatest specificty for G, then M then F

Question 17

What 2 methods are used to determine blood glucose?

Answer 17

1. Roche Assay
2. Glucometer

Question 18

Describe the methods in the Roche assay

Answer 18

• de-proteinise blood by adding 0.1ml to 1ml of perchloric acid.
• centrifuge for 15 mins at 3500rpm
• aspirate supernate layer and use as sample
• carry out proceedure
• record reults:
  *

Question 19

How is the concntration of glucose in sample calulated?

Answer 19

Conc in sample = (Absorbance sample/ absorbance standard) X 100

= mg glucose/ dl blood

to get to mmol/ L X 5.55

Question 20

How do glucometers work?

Answer 20

Stick impregnated with glucose oxygenase-peroxidase reagent

measure current produced by redox catalysed by GOD

Question 21

What is a problem with glucometer readings? what is a better alternative and wyh?

Answer 21

Only gives a snap shot of glucose levels

glycated haemoglobin - results from 6-8 weeks