ENZYME LEC 2 Flashcards
Cofactor?
Small molecule required by an enzyme to function
Coenzyme?
Cofactor- metal ions or organic molecules
Cosubstrate ?
cofactor which is Transiently associated with an enzyme
Prosthetic group
A cofactor which is associated with an enzyme
Holoenzyme
Is a cofactor enzyme complex which is active
Apoenzyme
a cofactor enzyme complex which is inactive
Cofactor consumption example
Vitamins
Lipid soluble vitamins ?
A and D
example In which coenzymes take place in catalysis in the citric acid cycle
thiamine pyrophosphate
Enzyme assay?
Way in which we can measure the rate of an enzyme controlled reaction
How can we measure the initial rate of a reaction?
product formation or substate disappearance
Rate units
micromol/min
enzyme unit?
1micromol/min (product)
Katal?
1mol/sec
Spectrophotometer? Example
Measure the light absorbance. Eg NAD+ absorbs at 340nm
discontinuous direct assay?
Stop reaction at particular points and measure either [P] OR [R]
Hardings test?
test for glucose
-reduces CuSO4 to Cu2O when boiled in alkaline solution
- Reaction of Cu2o with molybdate > green
Again use of spectrophotometer
Coupled assay issue with measuring enzyme activity?
Product from one reaction being used as a substate in the next reaction so it cannot be measured
direct assay
can measure rate of reaction (enzyme activity) through out, eg spectrophotometer volume of coloured solution formed being proportional to concentration present.
Coupled assay solution to problem?
2nd product measured. 2nd enzyme mist be in excess (so rate limiting step is the one to be measured)
Enzyme kinetics?
The effect certain reaction parameters have on a particular reaction
Hyperbolic reaction?
Is the rate relationship described by Micheal mentor
Parameters tested?- Enzyme kinetics ?
How sensitive a substrate ism how fast its metabolised
- Regulation of the enzyme?
Vo =
Initial rate of reaction
Micheal mentor time?
1913
Micheal menton equation
V= Vmax (s)/ Km[s]
Km value
the [s] in which the initial rate is Half its maximum
Assumptions made by Micheal Mentons model?
- S E+P step is irreversible
3. [ES] Is in a steady state
The greater the Km value?
The greater the enzymes affinity for the substrate
Kn?
Represents the rate constant for the nth step fo a reaction. Reverse is k-n
Km alters with
Temp and pH
K2?
Turner over number of Kcat
Vmax=..
equation
K2[Et]\Kcat[Et]
Units for Vmax, [S], Kcat
[P]/Time, Molar, time^-1
Carbonic anhydrase has a large
Kcat
Lineweaver-burk plot
Linnear Micheal mentons graph. 1 over everything
1/Vo= (Km/Vmax)(1/s) + 1/Vmax
Points:
1) 1/vmax
2) 1/-Km
3) Gradient?
x axis
y axis
1) y intersect
2) x intersect
3) Km/Vmax
x axis- 1[s]
y axis 1/Vo
think y= mx+c
Problems with lineweaver-burk plot?
1) Large values of [s] (most) are hard to detect (crowded on the left side of the graph as it 1/[s]
2) Small values of [s], small errors lead to large errors in 1/Vo and therefore large errors In Km and Vmax
Hanes-Woolf Plot
[s] plotted against [s]/Vo
Hanes-woolf plot equation
[S]/Vo= Km/Vmax +[S]/Vmax Mx= [S]/Vmax
