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Enzyme Kinetics

Based on the information given: a) In the absence of inhibitor: Vmax = 3 units/min Km = 2 mM b) In the presence of 2 mM inhibitor: Vmax decreases to 1.5 units/min Km increases to 4 mM c) This is competitive inhibition as Vmax remains the same but Km increases. The inhibitor constant (Ki) can be calculated from the change in Km and inhibitor concentration.

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89 views39 pages

Enzyme Kinetics

Based on the information given: a) In the absence of inhibitor: Vmax = 3 units/min Km = 2 mM b) In the presence of 2 mM inhibitor: Vmax decreases to 1.5 units/min Km increases to 4 mM c) This is competitive inhibition as Vmax remains the same but Km increases. The inhibitor constant (Ki) can be calculated from the change in Km and inhibitor concentration.

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jpm smurf
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© © All Rights Reserved
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Enzyme Kinetics

CHE182
Enzyme’s Lock and key theory
Enzyme’s Reaction Mechanism

• S + E ↔ ES Rapid/in equilibrium

• ES → P + E slow

• Apply steady state assumption (SSA)


• Apply rate determining state theory (RDS)
Michaelis-Menten Equation

• Where v is the velocity or rate of product formation


• vmax is maximum rate of product formation.
• [S] is the substrate concentration.
• KM is the Michaelis Menten Constant.
Sample Problem 1

In an experiment conducted to evaluate the Michaelis-


Menten constant, it was found that 1 g of bacteria could
decompose the waste at a maximum rate of 35 g/day when
the waste concentration was high. It was also found that
the same quantity of bacteria would decompose waste at a
rate of 18 g/day when the waste concentration was 20
mg/L. Calculate rate of waste decomposition by 2 g of
bacteria if the waste concentration were maintained at 8
mg/L.
A. 10.4 g/day B. 15.6 g/day C. 20.8 g/day D. 31.2 g/day
Quiz 5

1. _______ tags protein for destruction. 2pts


2. What vitamin helps in deamination of serine? 2
pts
3. What amino acid transport ammonia from
muscle?
4. Ornithine and Carbamoyl phosphate condensed to
form _______. 2 pts
5. The second ammonia of Urea is coming from this
amono acid. 2 pts
Lineweaver-Burk plot

y = mx + b
Sample Problem 2
Hanes-Woolf plot

y= b + mx
Eadie-Hofstee Plot equation.

y= b + mx
MM and Batch Reactors

=
MM and Batch Reactors

+
Sample Problem

• The initial concentration of glucose in a batch


reactor fermentation media is 100 g/L. Previous
rate study shows, at the same enzyme
concentration, that the maximum rate of
conversion of glucose to ethanol is 3 g/L.h and the
Michaelis-Menten constant is 2 g/L. Calculate the
number of hours it will take the yeast to reduce the
glucose concentration to 10 g/L.
CSTR and MM

• Fso – Fs = -rs V

• Where Fso is the initial substrate molar flow rate (moles of


solute/time)

• V is the volume of the reactor

• -rs is the Michaelis-Menten reaction rate


CSTR and MM

• τ = V/v

• Tau is residence time.

• Reactor volume/ volumetric flow rate.


CSTR and MM
CSTR and MM

• Consider a 10 L CSTR maintained at 310 K where


the initial glucose concentration is 1.00 moles/L
flowing at 1.00 L/s. Previous study shows that Vmax
is 1.2 moles/L.s and KM is 0.4 mol /L. Calculate the
final glucose concentration.
A continuously stirred tank reactor (CSTR) with a working
volume of 15 m3 is used for the production of baker's yeast.
The overall reaction for aerobic fermentation is expressed as:
0.556 C6H12O6 + 0.301 NH3 + 1.43 02 → 0.301C6H10O3N +
2.283 H2O + 1.532 CO
The empirical formula for the biomass is C6H10O3N (144) and
molasses contains 40.8% w/v glucose (180).Calculate the
daily molasses consumption to maintain a daily production of
one metric ton if the residual sugar in the outgoing broth is
1.40 % w/v glucose.

A. 1465L/day B. 2930 L/day


C. 4395 L/day D. 5860L/day
Specific Growth Rate, u

• u = 1/τ
• u = v/V
• The unit is per unit time.
Sample Problem

2. The specific growth rate of the yeast


is____
A. 0.13/day
B. 0.26/day
C. 0.39/day
D. 0.52/day
Sample Problem

• For a given species of a microorganism that


doubles every 3 h, what is the mass of biomass that
may be expected from 100 liters of seed if each
liter contains 8 grams biomass and the fermentation
culture was maintained for 24 h is____________
• A. 102 kg B. 204 kg
• C. 306 kg D. 408 kg
Quiz

• An aqueous solution of molasses contains 15% by


weight sucrose (C12H22O11). The CO2 formed in the
reaction can be considered as having a negligible
solubility in the solution. Determine the % wt
ethanol in solution after 95% of the sucrose has
been converted to ethyl alcohol by fermentation.
• A. 2.07% B. 4.14%
• C. 7.67% D. 8.27%
Old board problem
• Albizzia folcataria, a specie of plywood, after an initial
thermochemical hydrolysis yielded 25 % maltose, 3% sucrose,
12 % cellobiose, 43 % oligosaccharides and 17%non
carbohydrates residues. The resulting hydrolyzate is passed
through a column of immobilized enzyme systems so that all
types of dissaccharides are converted further to hexose units.
In alcohol fermentation, the rule of thumb is 10% of the
substrate is converted to biomass and 90% to alcohol. What
mass of ethanol is expected to be produced per six-day week if
one metric ton of pulpwood is processed daily?
• A. 1162kg B. 1223 kg
• C. 1885 kg D. 2526 kg
Quiz 2/Old board problem

One metric ton of wild potatoes were hydrolyzed


enzymatically and the hydrolyzate contained the following:
xylose 2% glucose 16% oligosaccharides 36% pentose 3%
maltose 20% others 23%.
It was observed that of the hydrolyzate, only maltose and
glucose were fermentable by alcohol yeast.
Calculate the mass of alcohol that can theoretically be
expected from the hydrolyzate.
• a. 189kg b. 95kg c. 338 kg d. 371kg
Plug Flow Reactor and MM
Plug Flow Reactor and MM
MM and Plug Flow Reactors

+
Sample Problem

• The initial concentration of glucose in a plug flow


reactor fermentation media is 100 g/L. Previous rate
study shows, at the same enzyme concentration, that
the maximum rate of conversion of glucose to
ethanol is 3 g/L.h and the Michaelis-Menten
constant is 2 g/L. Calculate the length of the plug
flow reactor needed if the diameter of the pipe use is
0.03 m to reduce the glucose concentration to 10 g/L
if the flow rate is 1000 L per day.
Competitive Inhibition
Competitive Inhibition

E + S → ES K1
E + I → EI
Therefore K1 decreases!

KM = (K2 + K3)/K1

Thus KM increases!
Competitive Inhibition

Vmax = K3 [E]t
K3 and [E]t are constant !
Therefore Vmax don’t
change.
Non-Competitive Inhibition
Non-competitive Inhibition

ES + I → ESI
Vmax = K3 [E]t
K3 decreases !
Therefore Vmax decreases.
Non-Competitive Inhibition

E + I → EI
E + S → ES
Therefore K1 decreases!
And since K3 also decreases
KM is unchanged!
Uncompetitive Inhibition
Uncompetitive Inhibition

ES + I → ESI
ES → E + P
Therefore K3 decreases
While K1 and K2 remain constant.
Therefore Vmax and KM decreases.

KM = (K2 + K3)/K1 Vmax = K3 [E]t


Eisenthal and Cornish-Bowden
Plot
The kinetics of an enzyme is measured in the
presence and absence of a 2 mM Inhibitor (I)

a. What are the values of Vmax and Km in the


absence of Inhibitor (I)? In its presence?
b. What type of inhibition is it?
The enzyme kinetics in the last problem is studied in the
presence of another Inhibitor. The concentration of this
inhibitor is 100 uM.

a. What are the values of Vmax and Km in the presence of


Inhibitor (I)?
b. What type of inhibition is it?

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