serine proteases Flashcards
why are ester substrates used to mimic a protein (amide) substrate for chymotrypsin in steady-state experiments?
reaction is easier to follow as product 1 has a yellow colour which can be followed in a spectrophotometer
why does a steady-state kinetics plot not continue through zero?
the pre-steady state phase prevents this - ES complex forms in ms before this can be detected
what is the rate determining step for serine proteases?
release of the second product
which 3 residues make up the catalytic triad for chymotrypsin?
serine, histidine & aspartate
how does the catalytic triad act?
- His acts as a base
- Ser acts as an acid
- Asp stabilises the positive charge on the His
what is the role of the oxyanion hole?
stabilises tetrahedral intermediate
why doesnt chymotrypsin digest itself?
it is synthesised as an inactive zymogen and activated after secretion by partial proteolysis
how does chymotrypsin differ from chymotrypsinogen?
chymotrypsinogen has a distorted conformation around Gly193 in the oxyanion hole. this prevents stabilisation of the transition state
why is the activity of serine proteases with the catalytic triad removed still more active than the uncatalysed reaction?
although catalytic activity has been removed, physical mechanisms of oxyanion hole stabilising transition state still present
why does hexokinase have a sequential mechanism?
- binds glucose then ATP to prevent water getting into the site
- in the absence of glucose, ATP reacts with water in a wasteful side reaction
why does lysozyme have a narrow pH range for optimal activity?
ionisation of catalytic residues can inactive them
why is the carbonium mechanism incorrect?
they suggested carbonium as an intermediate rather than a transition state
what did they do in the 2001 paper on the mechanism of lysozyme?
they made the E35Q glutamate -> glutamine mutant which had slowed rate
detected glycosylated lysozyme using mass spectrometry and X-ray crystallography
what are the purposes of serine proteases?
digestion blood coagulation blood pressure regulation blood clot lysis insulin production
what are the key features of serine proteases?
catalytic triad
oxyanion hole
substrate binding site
specificity pocket
