Enzymes 1 - Harmer Flashcards
3 main types of enzymes
- Kinases
- Transferases
- Isomerases
Ways of measuring enzyme reactions (5)
- Spectrophotometry
- Fluorescence (excited photons)
- Luminescence (photons emitted without E put in)
- FTIR (like IR)
- Synchotron radiation (EMF)
others: Radioactivity, mass spec, electrophoresis, HPLC,
Types of enzyme assays
- simple continuous assay
- stopped assay
- coupled assay
Continuous assays are good because
they provide lots of data points so we can work out the rate accurately from the initial slope - the steepest part (Vmax)
In a coupled assay, the intermediate formed by the enzyme of interest is then used
in a second reaction with a second enzyme and it is the product of the second reaction which is measured
In a coupled assay, the measurement of the rate is taken
where the line is steady state
(in a coupled assay, the initial rate is NOT used as it takes time for the intermediate to form for use in the second reaction - this is called the ‘lag phase’)
Shortening the time of the lag phase in a coupled assay can be achieved by
adding excess of second enzyme
The Michaelis Menten equation
Vmax = maximum rate achieved by the system, at saturating substrate concentration
Km = the Michaelis constant, the substrate concentration at which the reaction rate is half of Vmax
The purpose of collecting rate data is to
put it into the MM equation to determine the rate of an enzyme
A wide range of substrate concentrations is necessary for the MM equation to ensure
Vmax and Km are accurate (to determine the slope of the line)
What substrate concentrations should be used to define MM equation parameters?
3 2-fold dilutions above and below Km
