Enzyme Kinetics Flashcards
What is chymotrypsin?
What is chymotrypsin secreted by and in what form? How is this inactive form converted to active chymotrypsin?
What is the function of chymotrypsin?
Chymotrypsin - serine protease composed of 241 amino acids, arranged in 3 peptide chains (A, B, C) linked by disulphide bridges
Chymotrypsin is secreted by the pancreas as the pro-enzyme chymotrypsinogen. This inactive form undergoes proteolysis in the duodenum to form active chymotrypsin.
Chymotrypsin is an enzyme that can hydrolyse peptide bonds and aids protein digestion for absorption.
What are the requirements for a peptide to be recognised by chymotrypsin?
-aromatic side chain e.g. phenylalanine, tyrosine or tryptophan, with cleavage taking place on the carboxyl side of the peptide bond.
—> hydrophobic side chains
Why can we follow the hydrolysis of GNPNA to N-gluaryl-L-phenyl alanine and P-nitroaniline (bright yellow product), catalysed by chymotrypsin, via spectrophotometry?
What law does the absorption of p-nitroaniline obey?
How is the production of p-nitroaniline measured?
We can follow this reaction continuously by spectrophotometry since P-nitroaniline has significant absorbance at 410nm unlike GPNA.
The absorption of p-nitroaniline obeys the beer-lambert Law.
Production of p-nitroaniline is measured as a function of time using several diff concs of GPNA.
What are proteases key for regulation of?
What is the effect of migrating cells on the ECM?
Proteases are key for regulation of other enzymes
Degrade ECM by migrating cells
General protein turnover
What is Km known and defined as?
What is the Km value useful for biochemically?
What does a low and high Km indicate?
Km = Michaelis constant. Defined as the concentration of substrate at which a particular enzyme works at half its max velocity.
The km value is useful for comparing the strength of enzyme substrate complexes.
A low km -> indicates tight binding of a substrate to an enzyme
High Km -> indicates weak binding of a substrate to an enzyme.
At what stage of a reaction are enzyme substrate complexes formed and consumed at the same rate?
ESC formed and consumed at the same rate during the initial phase of the reaction, as long as the reaction velocity remains constant - reaction is in steady state.
Lineweaver burk plots:
What value does the y intercept represent on a lineweaver-burk plot?
What does the slope represent?
What value does extrapolating the straight line graph until it crosses the x-axis give?
Y intercept: 1/Vmax
Slope: Km/Vmax
X intercept = -1/Km
What happens to absorbance against time graphs as [S] increases?
How do you calculate V0 for higher substrate concs from absorbance against time graphs?
What are the units for KM?
The curves become less linear and tend to plateau.
For higher substrate concs you should calculate V0 for the initial linear part of the reaction. There can be some experimental errors in deciding which part of the data series is linear and therefore affects value for calc of V0.
Km units = units for substrate conc, as it’s graphically defined as the substrate conc at which half Vmax is achieved.
How can you tell using a lineweaver-burk plot whether enzyme inhibitors are competitive or non competitive?
What is the turnover of an enzyme? How it is calculated?
Competitive inhibitor: adding enough substrate will outcompete the antagonist. Competitive inhibitors will therefore inhibit Km but have no effect on Vmax.
Non competitive inhibitors: no effect of Km but act to lower Vmax.
Turnover of an enzyme (Kcat) = the number of molecules that an enzyme can process in a given unit of time, typically a second. Vmax/enzyme conc.
