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David J. Keffer
Department of Chemical and Biomolecular Engineering
The University of Tennessee, Knoxville
dkeffer@utk.edu
last updated: November 16, 2011
CH3CHO CH4 + CO
rate = k[CH3CHO]3/2
H2 + Br2 2HBr
3/ 2
k1CH 2 CBr
rate 2
CHBr k 2CBr2
A + M A* + M
Pseudo-Steady-State Hypothesis
nr
rA* ri , A* 0
i 1
1
example: decomposition of Azomethane (AZO)
(CH3)2N2 C2H6 + N2
mechanism
r1 k1C AZO
2
r3 k3C AZO*
There are no in or out terms. The PSSH dictates that the accumulation is zero, so we have
2
k1C AZO
C AZO*
k 2C AZO k3
2
k3k1C AZO
r3 k3C AZO*
k 2C AZO k3
2
At very low concentrations of AZO, k 2C AZO k3 , so the observed rate is
r3 k1C AZO
2
k3 k1
r3 C AZO
k2
In general
mechanism
reaction 1: A + M M + A*
reaction 2: A* + M A + M
reaction 3: A* P
r1 k1C ACM
r2 k2CM C A*
r3 k3C A*
k1C ACM
C A*
k 2CM k3
3
Because the concentration of all molecules (M) inert or otherwise is constant, we observe the rate
of production as
k3k1C ACM
r3 k3C A* kC A
k 2 CM k3
Chain Reactions
step 1: initiation
k1
C 2 H 6 2CH 3 r1 k1CC2 H 6
step 2: propagation:
k2
C2 H 6 CH 3 CH 4 C2 H 5 r2 k 2CC2 H 6 CCH
3
k3
C2 H 5 C2 H 4 H r3 k3CC H
2 5
k4
C2 H 6 H C2 H 5 H 2 r4 k 4CC2 H 6 C H
step 3: termination:
k5
2C2 H 5 C4 H 10 r5 k5CC H
2
2 5
nr
0 i , ri
i 1
4
nr
H: 0 i ,H ri 0 r1 0 r2 1 r3 1 r4 0 r5 r3 r4
i 1
nr
CH3: 0 i ,CH ri 1 r1 1 r2 0 r3 0 r4 0 r5 2r1 r2
3
i 1
nr
C2H5: 0 ri 0 r1 1 r2 1 r3 1 r4 2 r5 r2 r3 r4 r5
i ,C2 H 5
i 1
H: 0 k 3C C H k 4 C C 2 H 6 C H
2 5
Solve:
k1
CH3: CCH 2
3
k2
k3 CC2 H 5
H: CH
k 4 CC2 H 6
Solve
k1
C2H5: CC H 2 CC H
2 5
k5 2 6
k3 k1 1
H: CH 2
k4 k5 CC2 H 6
5
The rate of disappearance of ethane is
k1 CC2 H 6
r1 r2 r4 k1CC2 H 6 2k1CC2 H 6 k3 2
k 5 CC2 H 6
k1
r1 r2 r4 3k1CC2 H 6 k3 2 C C2 H 6
k5
k1
r3 k3CC H k3 2 CC H
2 5
k5 2 6
r5 k5CC H 2k1CC2 H 6
2
2 5
Reaction Pathways
Numerical Example
Provided below are two files that look at the same basic reaction using an active intermediate.
The first version of the file does not invoke the PSSH. The second version of the file does
invoke the PSSH. The purpose of including the files is several fold.
1. The first file, without using PSSH, shows that the same approach we have been using can be
used to model systems of reactions that result in non-elementary rate laws for the composite
reaction.
2. How one changes the calculations (or equivalently the code) to account for the PSSH.
3. The goodness of the PSSH is a function of the rate parameters, k1, k2 and k3. For the values
given, the results between the two versions of the code are similar. As one narrows the gap
between the slow reaction (k1) and the fast reactions (k2 and k3), one can see the goodness of
the PSSH approximation deteriorate.
6
Active Intermediate Example Input File without PSSH
%
% rate laws
%
k1 = 1.0e-3; % liters/mole/sec
r1 = k1*CA*CB; % mole/liter/sec
%
k2 = 1.0e-1; % liters/mole/sec
r2 = k2*CB*CC; % mole/liter/sec
%
k3 = 1.0e-0; % liters/mole/sec
r3 = k3*CC; % mole/liter/sec
%
% mole balances
%
dydt(1) = nuA1*r1 + nuA2*r2 + nuA3*r3;
dydt(2) = nuB1*r1 + nuB2*r2 + nuB3*r3;
dydt(3) = nuC1*r1 + nuC2*r2 + nuC3*r3;
dydt(4) = nuD1*r1 + nuD2*r2 + nuD3*r3;
7
Active Intermediate Example Input File with PSSH
8
Enzymatic Reaction Fundamentals
SP
ES ES EP
enzymes are specific. Typically, one enzyme catalyze only one type of reaction.
Therefore, unwanted by-products of side-reactions are usually avoided in enzymatic reactions.
Enzyme-Substrate Complex
enzyme have a specific active site which binds the substrate to form the enzyme-substrate
complex.
If the enzyme is exposed to extreme temperatures or extreme pH, it may unfold, losing its active
sites, becoming denatured.
example
substrate: S = urea
enzyme: E = urease
product : P = ammonia and carbon dioxide
9
reaction 2: reverse of reaction 1
k2
S E* S E r2 k 2C S E*
Et E S E *
nr
S E* : 0 i ,S E* ri 1 r1 1 r2 1 r3 r1 r2 r3
i 1
S E* : 0 k1C S C E k 2C S E* k3CS E* C H 2O
k1CS C E
S E* : C S E*
k 2 k3C H 2O
nr
dCS
S: i ,S ri 1 r1 1 r2 r1 r2
dt i 1
dCS
S: k1CS C E k 2C S E*
dt
dCS k1C S C E k2
S: k1C S C E k 2 k1 1CS C E
dt k 2 k 3 C H 2O k 2 k 3C H O
2
This expression is no good in a practical sense, since we can only measure the total enzyme
concentration.
k1CS CE
CEt C E CS E* C E
k 2 k3C H 2O
10
1 k 2 k3C H 2O
C E C Et C Et
1
k1CS k1CS k 2 k3C H 2O
k 2 k 3 C H 2O
dCS k2 k 2 k 3 C H 2O
k1 1 CC
dt k 2 k 3C H O k1CS k 2 k3C H O S Et
2 2
k1k3
CS C Et C H 2O
k1CS k 2 k3C H 2O
Michaelis-Menton Equation
The reaction is carried out in aqueous solution, so water is in excess and can be considered
constant.
kcat k3C H 2O
kcat k 2
KM
k1
Putting these new definitions into the balance for the substrate yields
dCS kcat
CS C Et
dt CS K M
The turnover number is given by k cat . This is the number of substrates converted to product per
unit time. For example, for the enzyme catalase with the substrate H2O2, the turnover number is
40x106 s-1. Forty million hydrogen peroxide molecules are decomposed by this enzyme every
second.
The constant KM is the Michaelis constant. It is a measure of the attraction of the enzyme for its
substrate. It is also called the affinity constant. For the example above, the Michaelis constant is
1.1 M.
If we define another variable, Vmax, which represent the maximum rate of reaction for a given
total enzyme concentration,
Vmax kcat C Et
We have
11
dCS V C
max S
dt CS K M
dCS V C
max S
dt KM
dCS V C
max S Vmax
dt CS
dCS V
Furthermore, K M is the concentration that yields max .
dt 2
dCS V C
max S
dt CS K M
Lineweaver-Burk plot is -1/rate vs 1/CS. Invert equation above. (See Figure E7-3.1) Straight
line.
1 C KM 1 K 1
S M
dC S Vmax C S Vmax Vmax C S
dt
So you can get Vmax from the intercept and Km from the slope.
12
If the generation of product is reversible:
Et E S E *
nr
S E* : 0 i ,S E* ri 1 r1 1 r2 1 r3 1 r4 r1 r2 r3 r4
i 1
S E* : 0 k1CS C E k 2C S E* k3C S E* C H 2O k 4C P C E
k1CS C E k 4C P C E
S E* : CS E*
k 2 k3C H 2O
nr
dCS
S: i ,S ri 1 r1 1 r2 r1 r2
dt i 1
dCS
S: k1CS C E k 2C S E*
dt
13
dCS k C C k 4C P C E k1k3C H 2O CS k 2 k 4C P
S: k1CS C E k 2 1 S E C
dt k 2 k 3 C H 2O k 2 k 3 C H 2O E
This expression is no good in a practical sense, since we can only measure the total enzyme
concentration.
k1CS C E k 4C P CE
C Et C E CS E* C E
k 2 k3C H 2O
1 k 2 k3CH 2O
CE C Et C Et
k1CS k 4C P k 2 k3C H 2O k1CS k 4CP
1
k 2 k3C H 2O
k1k3C H 2O CS k 2 k 4C P
CE
k 2 k3C H 2O k1CS k 4C P t
k cat k 3C H 2 O
k cat k 2
KM
k1
k4
kcat CS k 2
CP
dCS k1
C Et
dt k
K M CS 4 C P
k1
Vmax kcat C Et
14
k4 V
VmaxCS k 2 C P max
dCS k1 kcat
dt k4
K M CS C P
k1
k4
KP
k1 ratio of formation of enzymatic complex from substrate to that from product
Vmax
Vmax CS k 2 K P C P
dCS kcat
dt K M CS K P C P
kcat k k k k
KC cat 1 3 1 C H 2O
k2 K P k2 k4 k2 k4 ratio of many rates
Vmax CS P
C
dCS
K C
dt K M CS K P C P
(equation 7-29) of Fogler
In the simplest case, we have in a batch reactor a mole balance on the substrate
accumulation = generation
dCS
Sr
dt
dCS V C
max S
dt CS K M
CS K M
dCS Vmax dt
CS
15
1 1 V
dCS max dt
K M CS KM
1 1
CS t
Vmax
K M CS S K M t dt
CS ,o
dC
o
1
CS CS ,o ln CS Vmax t to
KM C S ,o KM
This expression relates reactor time to substrate concentration. It can not be analytically solved
for the substrate concentration.
16
Inhibition of Enzyme Reactions
Inhibitors are a molecule or species that renders the enzyme unable to catalyze a specific
reaction. In biological systems, some inhibitors have negative effects (e.g. cyanide acts an
inhibitor to a single enzyme in the aerobic oxidation process that will lead to death) and positive
effects (e.g. in the treatement of leukemia). Aspirin inhibits enzyme that catalyzes the synthesis
of a compound involved in the pain-producing process.
Competitive Inhibition
The rate of the formation of the product is still the rate of reaction 3.
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
enzyme complex.
nr
SE: 0 i ,S E ri 1 r1 1 r2 1 r3 r1 r2 r3
i 1
17
Solve for the concentration of the enzymatic complex
k1CS C E k 2 k3C H 2O
SE: C S E or CE CS E
k 2 k3C H 2O k1CS
The concentrations of the inhibitor-enzyme complex is unaffected by the substrate.
nr
I E: 0 i ,I E ri 1 r4 1 r5 r4 r5
i 1
I E: 0 k 4 C I C E k 5C I E
k 4C I C E
I E: C I E
k5
Et ES E I E
k 4C I C E kC
C Et C E C S E C S E 1 4 I C E
k5 k5
k C k 2 k 3 C H 2O kC k 2 k 3 C H 2O
C S E 1 4 I C S E 1 1 4 I C S E
k5 k1C S k5 k1CS
1
r3 k3C S E C H 2O k3C H 2O C Et
k C k 2 k 3 C H 2O
1 1 4 I
k5 k1C S
kcat k3C H 2O
1
r3 k cat C Et
kC k 2 k cat
1 1 4 I
k5 k1C S
18
kcat k 2
KM Vmax kcat C Et
k1 and
1
r3 Vmax
kC KM
1 1 4 I
k5 CS
k5
KI
k4
Vmax C S
r3
C
C S 1 I K M
KI
C
K M' 1 I K M
KI
Uncompetitive Inhibition
The rate of the formation of the product is still the rate of reaction 3.
19
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
substrate-enzyme complex.
nr
SE: 0 i ,S E ri r1 r2 r3 r4 r5
i 1
nr
I ES: 0 i ,I ES ri r4 r5 k 4C I CS E k5C I ES
i 1
k4
I ES: C I E S C I CS E
k5
k4
SE: 0 k1CS C E k 2CS E k3CS E k 4C I CS E k5 C I CS E
k5
Simplify
k 2 k3
SE: CE CS E
k1CS
C Et C E C S E C I E S
k 2 k3 k k k3 k 4
C Et C S E C S E 4 C I C S E 1 2 C I C S E
k1C S k5 k1C S k5
1
r3 k3CS E k3 C Et
k 2 k3 k 4
1 CI
k1CS k5
20
k3 k 2 k5
KM Vmax k3C Et KI
k1 and and k4
Vmax Vmax C S
r3
K
1 M I
C C
K M 1 I C S
CS K I KI
Noncompetitive Inhibition
The rate of the formation of the product is still the rate of reaction 3.
21
We use the PSSH to determine the concentrations of both the substrate-enzyme and inhibitor-
enzyme and inhibitor-substrate enzyme complexes.
nr
SE: 0 i ,S E ri r1 r2 r3 r6 r7
i 1
nr
I E: 0 i ,I E ri r4 r5 r8 r9 k 4C I C E k5C I E k8CS C I E k9C I ES
i 1
nr
I ES: 0 i ,I ES ri r6 r7 r8 r9 k6C I CS E k7 C I ES k8CS C I E k9C I ES
i 1
k 4 C I C E k9 C I E S
I E: CI E
k 5 k8 C S
k 4 C I C E k9 C I E S
I ES: 0 k 6 C I C S E k 7 C I E S k8C S k9 C I E S
k 5 k8C S
k 4 k8C S C I C E
k6C I C S E
k 5 k8 C S
C I E S
kkC
k 7 9 8 S k9
k 5 k8C S
k 4 k8C S C I C E
k6C I C S E
k 5 k8 C S
SE: 0 k1C S C E k 2CS E k3CS E k6C I CS E k 7
kkC
k7 9 8 S k9
k 5 k8C S
Simplify
k 7 k 4 k 8C S C I
k k k C k 7 k6C I C k C k 5 k8C S C
SE:
2 3 6 I
k 9 k 8C S S E 1 S k 9 k8C S E
k k 9 k k 9
k 5 k8C S k 5 k8C S
7 7
22
k k k C k 7 k6C I
2 3 6 I
kkC
k 7 9 8 S k9
k 5 k8C S
SE: CE C S E
k 7 k 4 k 8C S C I
k 5 k 8C S
k1CS
kkC
k 7 9 8 S k9
k 5 k 8C S
Simplify
C Et C E C S E C I E C I ES
r3 k3C S E
Vmax C S
r3
End Result from Fogler. Noncompetitive
K M CS 1 C I
KI
Compare with:
Vmax C S
r3
C Competitive
C S 1 I K M
KI
Vmax C S
r3
C Uncompetitive
K M 1 I CS
KI
23
Substrate Inhibition: In any of these cases, the inhibitor could be the substrate. Substitute S for I
in these results for rate law.
Enzyme co-factors: In many enzymatic reactions, you need two substrates. A rate law could be
derived following these same procedures.
24
Bioreactors
A bioreactor is a reactor that sustains and supports life for cells and tissue cultures.
reaction
rg CC
CS
max
K S CS
CS
rg maxCC
K S CS
Compare to the Michaelis-Menten equation for enzymatic reaction rates without inhibition.
dC S V C
max S
dt K M CS
In the case of many substrates, the concentration of the limiting substrate is used.
CS
rg k obs max CC
K S CS
25
n
C
where kobs 1 *p ,
C
p
C p is concentration of product
C *p is concentration of product where metabolism ceases
n is an empirical constant.
C
rg maxCC 1 exp S Tessier equation
k
1
rg max CC
Moser equation
1 kCS
rd k d kt Ct CC
Stoichiometry
1
Yc / s
Ys / c
Product formation can take place during different phases of the cell growth cycle.
26
Product formation during the exponential growth phase
CS
rp Y p / c rg Yp / c CC Y p / c max CC
K S CS
Product formation during the stationary phase where no cell growth occurs, we relate product
growth to substrate consumption
rp Y p / s rs
Part of the substrate must be used to maintain a cells daily activities: maintenance utilization
term
rsm mCC
If it is possible to sort out the S consumed for cell growth and S consumed for P then
S Yc/ s C + Y p / s P
substrate utilization
27
balance
net generation = rate consumed by cells + rate consumed to form product + rate consumed for
maintenance
During the growth phase, you may not be able to separate purpose of substrate consumption
rs Ys / c rg mCC
rp Y p / c rg
During stationary phase, growth is zero and these equations dont work
CSn
rp k p CC (Monod equation)
K Sn CSn
Sn = secondary nutrient
CSn
rSn YSn / p rp mCC YSn / p k p CC mCC
K Sn CSn
28
Mass Balances
Cell balance
substrate balance
dC S
V Fin C S ,in Fout C S Vrs
dt
Cell Balance
V rg rd
dCC
V
dt
Substrate balance
growth phase
dC S
V Vrs VYs / c rg VmCC
dt
stationary phase
dC S
V Vrs VYs / p rp VmCC
dt
product
dC P
V Vrp VY p / s rs (seems obviously incorrect since some substrate is used for
dt
maintenance, not production)
29
This seems better:
dC P
V Vrp VY p / s rs (seems obviously incorrect since )
dt
Other notes:
dilution rate
F 1
D
V R
dCC
cells: 0 DCC ,in DCC rg rd
dt
dC S
substrate: 0 DC S ,in DC S rs
dt
dC P
substrate: 0 DC P ,in DC P rp
dt
30