Q.1 The following data were recorded for the enzyme catalyzed reaction S P.

[S] (M) v(nmoles/litre min)

6.25×10−6 15.0
7.50×10−5 56.25
1.00×10−4 60
1.00×10−3 74.9
1.00×10−2 75
a)Estimate Vmax and Km
b)what would be v at [S]= 2.5×10−5 M and [S]= 5×10−5 M.
c)what would v be at [S]= 5×10−5 M if enzyme concentration is doubled.

Q.2 An enzyme was assayed /evaluated at an initial substrate concentration of 10−5 M. the Km for
the substrate is 2.00×10−3 M at the end of 1 min 2% of substrate had been converted to product.
(a)what percent of substrate will be converted to product at the end of 3min? (b) If initial concentration
of substrate were 10−6 M what percentage of substrate will be converted to product after 3min. (c)
What is the maximum attainable velocity(Vmax) with the enzyme concentration used.

Q.3 The following data were obtained for an enzyme that catalyses the reaction S P. The substrate
concentration have been spaced to allow use of any of linear plots . Plot the data and determine Km and
Vmax.
[S] (M) v(nmoles/litre min)
8.33×10−6 13.8
1.00×10−5 16.0
1.25×10−5 19.0
1.67×10−5 23.6
2.00×10−5 26.7
2.50×10−5 30.0
3.33×10−5 36.3
4.00×10−5 40.0
5.00×10−5 44.4
6.00×10−5 48.0
8.00×10−5 53.4
1.00×10−4 57.1
2.00×10−4 66.7
Q.4 In some enzyme catalyzed reactions, multiple complexes are involved as follows:
K1
S+E (ES)1
K2
K3
(ES)1 (ES)2
K4

K5
(ES)2 P+E

Develop a rate expression using (a) the Michaelis-Menten approach and (b) the briggs-Haldane
approach.

Q.5 The michaelis-Menten approach assumes that the product releasing step is much sloer than the
first complex forming step of the simple enzyme reaction mechanism:
K1
S+E (ES)
K2
K3
(ES) E+P
derive a rate equation for the case in which the enzyme substrate formation step is much slower than
the product releasing step, that is, K1<<K3,K2<<K1. State your assumption.

Q.6 Yang and Okos (1989) proposed an alternative kinetic model of lactose hydrolysis by Aspergillus
niger lactase:
K1 K3
S+E ES EP+Q
K2

K4
EP E+P
K55
where S.P.Q and E are lactose, galactose, glucose and free enzyme. In this mechanism, Glucose is
released from the enzyme-substrate complex first. Leaving the enzyme-galactose complex which
subsequently releases the galactose molecules.
A) Derive the rate equation for the production of galactose by using Briggs-Haldane approach.
B) How is the rate equation developed by this model simplified to Scott et al.(1985) in the previous
problem?

Q.7 The value of Km and rmax for an enzyme (21 degree C and pH=7.1) are 0.004Kmol/ m3 and 10
Kmol/sm3 respectively. We immobilized this enzyme by attacking it covalently to acrylamide-based
polymers that can be assumed to have spherical shape (diameter=1mm). The effectiveness of the
immobilized enzyme was found to be 70 percent of the free enzyme when the concentration of the
substrate was 0.5 Kmol/m3 . The reaction was carried out in a stirred reactor with an agitation speed
of 50rpm.
a) estimate the concentration of the substrate at the surface of the immobilized enzyme.
b) estimate the mass transfer coefficient in cm/sec.

Q.8 An enzyme is immobilized on the solid surface. Assume that the external mass-transfer resistance
for the substrate is not negligible and that the michaelis-Menten equation describes the instrinsic
kinetics of enzyme reaction.
a) Derive an expression which shows the relationship between the substrate concentration at the surface
and that in the bulk solution.
b) the following equation was found to fit the data for the immobilization enzyme reaction,

Csb (rmax)app
robs=
(km) app +C sb
Do you expect that the values of rmax app and Kmapp are similar to rmax and Km for the free
soluble enzyme? If they are different what will be the meaning of rmax app and Kmapp ?

Q.9 Invertase is immobilised in ion-exchange resin of average diameter 1 mm. The amount of enzyme
in the beads is measured by protein assay as 0.05 kg m−3 . 20 cm 3 beads are packed into a small
column reactor; 75 ml sucrose solution at a concentration of 16 mM is pumped rapidly through the bed.
In another reactor an identical quantity of free enzyme is mixed into the same volume of sucrose
solution. Assume the kinetic parameters for free and immobilised enzyme are equal: K m is 8.8 mM
and the turnover number is 2.4×10−3 gmol glucose /(g enzyme)s. The effective diffusivity of
sucrose in the ion-exchange resin is 2×10−6 c m 2 s -1.
(a) What is the rate of reaction by free enzyme?
(b) What is the rate of reaction by immobilised enzyme?