BIOC2302 Chymotrypsin kinetics

what type of enzume is chymotrypsin

serine protease (prod in pancreas) that hydrolyses peptide bonds

what residues make up the catalytic triad

serine histidine aspartic acid

serine protease catalytic mechanism

generate acyl-enzyme intermediate that is slowly converted back to the active enzyme (deacetylation)

how does the catalytic mechanism of serine proteases enable determining number of active sites

burst phase of the reaction is very fast (prod acyl-enzyme intermediate) where the deacetylation step is much slower. so burst phase can be used to determine number of active sites per enzyme.

will serine proteases also hydrolyse esters as well as proteins

yes

what substrate was used in the chymotrypsin kinetics experiment

4-nitrophenyl acetate (ester of acetic/ethanoic acid)

why is using 4-nitrophenyl acetate useful in studying chymotrypsin kinetics

one of products is 4-nitrophenol, (in 4-nitrophenolate form (ionic form)) absorbs strongly. Maximum abs. at 405 nm

what wavelength does 4-nitrophenol (4-nitrophenolate) absorb most strongly

405 nm

colour of 4-nitrophenol (ate)

yellow

what is the fast (burst) phase of the reaction

acetyl group transferred from 4-nitrophenyl acetate transferred to the enzyme, releasing 4-nitrophenol (yellow)

what is the slow stage of the reaction

removal of the acetyl group via hydrolysis, to restore original form of enzyme (prod acetic acid)

how to measure burst phase of the reaction

will observe a linear increase in absorbance at 400 nm (reached steady state), but extrapolate back to t=0 and it wont cross at the origin. this represents the burst of 4-nitrophenol release. (too quick to measure with spectrophotometer)

If 10 microM 4-nitrophenolate at t=0 was measured for 10 microM chymotrypsin then how many active sites are there per enzyme

1

If you found 20 microM of 4-nitrophenolate was produced in the burst phase with 10 microM chymotryspin, how many active sites does each trypsin molecule have

2 active sites

define specific activity

the amount of reactant (nmol) converted per minute per mg of enzyme

what plot has a gradient equal to specific activity

activity (measured rate) vs enzyme concentration (giving those activities)

why is the chymotrypsin catalysed reaction performed at pH 7.6 (pI = 7.0)

so the 4-nitrophenol product is in the 4-nitrophenolate form (anion) which has the yellow colour

is 4-nitrophenyl acetate soluble in water

NO insoluble in water, but soluble in propan-2-ol so its used at 5% in phosphate buffer

purpose of an absorbance - 4-nitrophenolate concentration calibration curve

enable conversion of abs into concentration (using the abs of known conc sols)

can det the molar extinction coefficient (M-1cm-1)

what is the formula mass of 4-nitrophenol

139.1 da

waht is the UV range

100- 400 nm

which cuvettes are uv transmissible

quartz cuvette (glass abs UV light)

mr of chymotryspin

23000 Da

does 4-nitrophenyl acetate slowly hydrolyse in the phosphate buffer used

yes it does, its why stock substrate is added to the reaction mix, so only contacts the buffer in the reaction mix)

can perform a control run without enzyme, to find how much this effect is when in reaction mix, so can account for it