enzyme kinetics

Question 1

chymotrypsin

Answer 1

241 AA
3 peptide chains joined by disulphide bridges
secreted by the pancreas as proenzyme chymotrypsinogen
inactive form undergoes proteolysis in duodenum = chymotrypsin - hydrolyse peptide bonds and aid digestion

Question 2

what does chymotrypsin recognise

Answer 2

aromatic side chain eg phenylalanine, tyrosine or tryptophan
cleavage on carboxyl side of peptide bond

Question 3

reaction with chymotrypsin

Answer 3

N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) hydrolysed to N-glutaryl-L-phenyl alanine and p-nitroaniline (yellow)

Question 4

how do we measure the reaction

Answer 4

continuously by spectrometry - p-nitoaniline significant absorbance at 410nm
follows the beer lambert law

Question 5

E for p-nitroaniline

Answer 5

8.8umol/ml/cm

Question 6

how do you measure production of p-nitroaniline

Answer 6

as a function of time

diff conc of substrate

Question 7

rate of enzyme catalysis

Answer 7

Michaelis and menten

Vo = Vmax([S]/[S]+Km)

Question 8

Vo

Answer 8

initial rate of rn

can be determined graphically by plotting rate oif product formation against time

Question 9

Vmax

Answer 9

max enzyme velocity

Question 10

Km

Answer 10

Michaelis constant

substrate conc where rate is half of Vmax

Question 11

what is allowed by GPNA being in excess

Answer 11

steady state reaction phase conforms to M and M kinetics

can be analysed by Lineweaver Burk plot

Question 12

why is chymotrypsin kept on ice

Answer 12

lose activity at room temp

Question 13

reagents for rn

Answer 13

buffer pH 7.8
20mM GPNA
20mg/ml chymotrypsin

Question 14

protocol for the investigation

Answer 14

spectrophotometer at 410nm 
pipette varying amounts of GPNA and buffer into cuvettes to create a range of substrate conc 
start at highest conc GPNA 
mix contents by inverting twice
calibrate spectrophotometer using this 
add 50ul of enxyme soln 
mix
record abs at 30sec intervals

Question 15

max vol given by P1000 pipette

Answer 15

1ml

Question 16

how do you pipette 50ul

Answer 16

P100 pipette yellow

set to 050

Question 17

Lineweaver-Burk plot

Answer 17

x axis - 1/[s]
y axis - 1/Vo 
gradient = Km/Vmax 
Vo calculated for range of [s] 
as [s] increases 1/[s] tends to 0 
1/Vmax = 1/Vo at y axis when 1/[s] = 0 
extrapolate line to x axis 1/[s] = -1/Km

Question 18

effect of competitive inhibitor on Vmax and Km

Answer 18

Vmax same

Km increased

Question 19

effect of non-comp inhibitor

Answer 19

Vmax less

Km same

Question 20

why does initial rate have to be measured

Answer 20

rate decreases with time at high conc - need to be steady states kinetics where enz-sub complex is made and consumed at equivalent rates for the Michalis menton kinetics to work

Question 21

forming a lineweaver burk plot

Answer 21

calculate change in absorbance from abs time graph
use A = ecl to calculate change in conc
then can plot L-B

Question 22

errors in the L-B graph

Answer 22

error determining the intercepts
magnified because reciprocals are taken especially at lower conc where little product was produced as only have a few points or had outliers

Question 23

turnover number

Answer 23

max number of molecules that can be converted into product per catalytic site at given conc per unit time

Question 24

comparison of chymotrypsin, carbonic anhydrase and lysozyme

Answer 24

carbonic anhydrase - active all the time higher turnover
chymotrypsin only in digestion
lysozyme - antimicrobial