BMP: Enzyme specificity Flashcards
What is the active site?
The three dimentional catalystic site at which enzyme-substrate interaction occurs
What 2 models are used to describe enzyme-substrate interaction?
- Lock and key - The enzyme and substrate are shaped to fit perfectly
- Induced fit - active site and substrate alter shape to connect
Compare the structures of mannose. glucose and fructose
Why is sodium fluoride added to the blood sample?
TO prevent glycolysis - prevent break down of glucose
What is normal resting blood glucose levels?
70-100 mg/dl
What is an example of a condition where blood glucose levels differ?
Diabetes melitus
What is glycated haemoglobin (HbA1c) and what is the desirable range for healthy adults?
when haemoglobin joins to glucose in the blood
below 42mmol/L
What is substrate specificty determined by?
- Structure
- Charge
- Polarity
- hydrophobicity
of the substrate binding molecule
Give an example of a highly specific enzyme and unspecific enzyme and its substrates
Specific: Ureases - urea
unspecific: serine proteases
What is the enzyme used in this experiement to demonstrate enzyme specificity
Glucose oxygenase
- What type of protein is glucose oxygenase?
- What is its molecular weight?
- What cofactor is attached?
- Dimeric protein
- 160kDa
- flavin adenine dinucleotide to each monomer
Describe the oxidation of glucose
- B-D-Glucose + O2 —(glucose oxygenase)–> 5-lactone + H2O2 + B-D-glucono-1
- 5-Lactone + H2O2 —(H20)–> D-gluconic acid + H2O2
- 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
What is the enzyme solution made off?
phosphate buffer (pH7), Peroxidase, Glucose oxidase (GOD), ABTS
What is the blank made off and whta wavelength is it zeroed at?
- 0.2ml water and 5ml enzyme solution
- 436nm
Describe the procedure to determine enzyme specificty
- Substrate solution (0.005M of M,F,G) and enzyme solution added together. Transfer sample directly to spectophotomoeter for zero reading.
- Repeat every minute for 10 minutes.
- Repeat for each substrate and ensure blank redone each time
- PLot graph of absorbance against time for 3 substrates
- Calculate the gradient for each substrate (linear section) and determine 7oxidation (assume O2 100%)
Rate oxidation of the sugars from highest to lowest. What can be said about enzyme spcificity?
G, M, F
Glucose oxygenase have greatest specificty for G, then M then F
What 2 methods are used to determine blood glucose?
- Roche Assay
- Glucometer
Describe the methods in the Roche assay
- 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:
*
How is the concntration of glucose in sample calulated?
Conc in sample = (Absorbance sample/ absorbance standard) X 100
= mg glucose/ dl blood
to get to mmol/ L X 5.55
How do glucometers work?
Stick impregnated with glucose oxygenase-peroxidase reagent
measure current produced by redox catalysed by GOD
What is a problem with glucometer readings? what is a better alternative and wyh?
Only gives a snap shot of glucose levels
glycated haemoglobin - results from 6-8 weeks
