Enzyme Kinetics Flashcards
What is chymotrypsin?
Its a serine protease
Secreted by the pancreas as pro-enzyme chymotrypsinogen
This inactive form undergoes proteolysis in the duodenum to form active chymotrypsin which can hydrolyse peptide bonds and aid protein digestion
What are requirements for the recognition of chymotrypsin?
An aromatic side chain such as phenylalanine, tyrosine or tryptophan
Chymotrypsin cleavages proteins on the carboxyl side of the peptide bond
What does chymotrypsin do?
Catalyses the hydrolysis of GPNA generating N-Glutaryl-L-phenylalanine and bright yellow product, P-nitroalanine
What does absorption spectra of P-Nitroalanine look like?
Follows the beer-lambert law with molar absorption coefficient of 8.8 micro mol ml cm^-1
What is Km?
Michaelis constant
Its the concentration of a substrate at which a particular enzyme works at half its maximal velocity
Km is useful for comparing the strength of enzyme-substrate complexes
Low Km indicates tight binding of substrate to enzyme
How do you calculate Vmax?
V0 is the initial velocity so v0 = Kcat [ES]
The max velocity occurs when emzyme is saturated- all enzymes are tied up with substrate
[ES] = [E[total
Vmax = Kcat [E]total
Kcat = turnover number
How is a steady state achieved?
In the initial phase, as long as velocity remains constant, reaction is in the steady state- ES is formed and consumed at the same rate
What is a lineweaver- Burk plot?
A double reciprocal plot of 1/V0 (y) against 1/[S] (x)
The y axis intercept is 1/Vmax
The x intercept is -1/Km
The slope os Km/Vmax
How do you calculate [GPNA] from stock concentration?
V1C1 = V2C2
V1= volume of GPNA stock C1= moles of GPNA V2= total volume C2= concentration of GPNA
What is the equation for absorbance?
A=ELC
Where
A= absorbance of solution
E= Extinction coefficient, a constant for the substance at a particular wavelength
C= conc. of the absorbing substance (mol/L)
L= path length in cm
How would you plot a lineweaver- Burk plot?
1) Plot a graph of Substance concentration against absorbance at indicated time
2) calculate the gradient for each time interval to find change in absorbance per second
3) calculate change in absorbance per minute by multiplying last answer by 60
4) Find [S] per min by using the equation A=ELC, to do this divide your previous answer by E (this is given to you in instructions). This is V0
5) to find 1/ [S] divide 1 by the concentrations in your table
6) Plot a graph of 1/[S] by 1/V0 and draw a line of best fit. Extrapolate to find your x and y intercept
How can a Lineweaver-Burk plot be used to assess mechanism of drugs?
Can be used to see if an enzyme is competitive or non-competitive
What does a lineweaver-burk plot look like for a competitive inhibitor?
No effect on Vmax
Km is greater (x intercept will be more positive)
What does a lineweaver-burk plot look like for a non- competitive inhibitor?
No effect on Km
Lower Vmax
What is turnover number?
The number of molecules an enzyme can process in a given unit of time, typically a second
