Enzyme Kinetics Flashcards
What is chymotrypsin?
Enzyme, consists of 241 amino acids
3 chains (A,B,C) linked by disulphide bridges
Secreted by the pancreas as the proenzyme chymotrypsinogen. This undergoes proteolysis in the duodenum to form chymotrypsin.
The enzyme hydrolysed peptide bonds and aids in protein digestion
In officer for chymotrypsin to be recognised the polypeptides being broken down must have an aromatic side chain
What are three amino acids that have an aromatic side chain?
Tryptophan
Phenylalanine
Tyrosine
Chymotrypsin reacts with the artificial substrate N-glutaryl-L-phenylalanine p-nitroanilide (GPNA). What does this reaction form?
Hydrolysis forms:
N-glutaryl-L-phenylalanine as well as the bright yellow product p-nitroaniline
How is the reaction between chymotrypsin and GPNA followed?
Continuously by spectrophotometry, since p-nitroaniline has a significant absorbable at 410 nm unlike GPNA
The absorption obeys the beer-lambert law, with a molar absorbtion coefficient of 8.8 micro mol ml cm^-1
What are some features of chymotrypsin?
Serine protease (at the core of its active site it has a serine)
Also useful for the regulation of other enzymes, eg their activation
As well as the degradation of the ECM by migrating cells
What is the specificity of chtymotrypsin?
Bulky hydrophobic side chains such as aromatics (tyrosine, phenylalanine and tryptophan) and also on methionine
What is Km?
Michaelis constant
Defined as the concentration of substrate for which any enzyme is working at half it’s maximal velocity
Useful for comparing the strength of enzyme substrate complexes
Low: tight binding of a substrate to the enzyme
High: weak binding
How are Vmax and Km calculated (equation and assumption)
V0 = Vmax[S]/(Km + [S])
V0 is needed as during the initial stage of the reaction the velocity remains constant as it is in a steady state. ES complexes are being formed and consumed at the same rate
What is a lineweaver-burk plot?
Double reciprocal plot of 1/V0 (y)
Against 1/[S] (x)
The y intercept gives 1/Vmax
The x intercept gives -1/Km
The gradient is Km/Vmax
How do competitive and non competitive inhibitors effect the lineweaver-burk plot?
Non competitive - same x intercepts but different y intercept
Competitive - same y intercept (Vmax) butbdofferent x intercept
What is the turnover number of an enzyme?
(Kcat)
The number of molecules an enzyme can process in a given unit of time
If the enzyme conc is known than it is simply calculated by Vmax/[enzyme]
