CHE 436: BIOCHEMICAL ENGINEERING

LECTURE 1:
ENZYME KINETICS

Lecturer:
ENGR. MICHAEL ALLAN G. RAMOS
Department of Chemical Engineering
Technological Institute of the Philippines
 ENZYMES

- Proteins of high molecular weight. 12,000 to 106 Da or g/mole.

Needs to be in a tertiary structure to function/ have catalytic activity.
Protein-folding/ denaturation vice versa

- Act as biological catalysts which results in higher reaction rates than

chemically catalyzed reactions under ambient conditions

- Naming: -ase, -in, depends on type of rxn, substrate

- Lowers activation energy of the reaction by binding the substrate

and forming an enzyme-substrate complex.
 SOURCE OF ENZYMES
- Microorganisms as host cells, protein expression

- Not just their own proteins but also recombinants (foreign genes
inserted into cells using a vector to produce a desire protein)

- i.e. Bacteria, plants cells and animals cells can be hosts for protein
synthesis using a vector for replication such as a virus

- cells are harvested, lysed and enzymes are extracted

- 80% of their value is on industrial process biocatalysts

ENZYMATIC CATALYSIS
MEDICINAL USES OF ENZYMES
 ENZYME KINETICS
Enzyme kinetics – quantitative analysis of the effect of various parameters on the rate of
enzyme catalyzed reactions. It involves molecular dynamics and requires experimental
data.

Why do we do kinetics?
- characterize enzymes and catalytic mechanisms in the reaction

- to get optimum kinetic parameters, km’, vm in running the reaction (concentration, pH,
temp) for large-scale production especially in estimating the time of reaction

- to design and operate bioreactors since kinetic study will help choose type of reactor

- screen different enzymes to be used for production

- detect inhibitions which is very important in drug discovery

 ENZYME KINETICS

- Starts with hypothesizing a suitable rate equation (recall rate equations for
elementary and non-elementary reactions)

- conduct experiment to validate the rate equation

- change rate equation/ hypothesis if data does not confirm

Rate equation

Experimental data Mechanistic hypothesis

HOW ENZYMES WORKS?
ENZYME KINETICS
ENZYME KINETICS
 MICHAELIS-MENTEN KINETICS

Derive M-M equation for Quasi steady-state assumption

 MICHAELIS-MENTEN KINETICS

How does the substrate concentration affect the order of the reaction?
 TURN-OVER NUMBER

k 2 = kcat = turnover number = no. of substrate molecules

converted to product per enzyme molecule per unit of time, when
[E] is saturated with substrate
BATCH EXPERIMENT
PLOTTING EXPERIMENTAL DATA

-good for low [S]

-for evaluating inhibitions
 PLOTTING EXPERIMENTAL DATA

-Large errors but less bias for low [S]

-For accurate det’m of vm

Note the y-axis for all plots to aid derivation

 INTERPRETATION OF Km’ and vm

Catalytic efficiency
k2
= the higher the ratio, the higher the specificity, the faster it binds
K m'

Fractional saturation
v [E .S ]
= = occupied sites / total active sites
vm [E0 ]

Km’ = depends on rate parameters and will change with changes with
temperature and pH; low value means tight binding, high value means
weak binding

vm = function of rate constant k2

CATALYTIC EFFICIENCY
COMPLEX ENZYME KINETICS: INHIBITION

- Analogues of substrate binds to E only and competes with S

Note all equations for inhibitions somewhat resembles original M-M

equation
COMPLEX ENZYME KINETICS: INHIBITION
 COMPLEX ENZYME KINETICS: INHIBITION

C) Uncompetitive Inhibition

- Inhibits binding to ES complex only and no affinity for the enzyme

k2
vm ,app .[S ] S S
v=
k m' ,app + [S ]
KI

Where;
vm k m'
vm ,app =
æ
çç 1 +
[I]ö
÷÷
km' ,app =
æ [I]ö
çç 1 + ÷÷
è KI ø è KI ø
 COMPLEX ENZYME KINETICS: INHIBITION

D) Substrate Inhibition
k2
vm .[S ] S S
v=
k m' + [S ] +
[S]
2

KS S
KS

S2
[Smax]
How to calculate for [Smax]?

dv
=0
d [S ]

[Smax ] = km' .k s
SUMMARY OF INHIBITIONS
EFFECT OF TEMPERATURE
 EFFECT OF pH

Optimal pH should be between pK1 – pK2

v=
vm .[S ] é K2
= km' .ê1 + + +
[ ]
H+ ù
k m' ,app + [S ]
km' ,app
ë [ ]
H K ú
1 û
 ILLUSTRATIVE EXAMPLE

An enzyme was assayed at an initial substrate concentration of 10-5 M. Km’ for the
substrate is 2x10-3 M. At the end of 1 minute, 2% of the substrate had been converted to
product.

(a) What percent of the substrate will be converted to product at the end of 3 minutes?
Calculate product and substrate concentration.

(b)If 10-6 is used, what percentage of the substrate will be converted to product after 3
minutes?

(c)What is the maximum attainable velocity of the reaction with the enzyme
concentration used?

(d)At what substrate concentration will vm be observed?

(e)At this substrate concentration, what percentage of substrate will be converted after
3 minutes?
 ILLUSTRATIVE EXAMPLE

Aspartic acid is produced by an enzymatic reaction. Aspartase, the enzyme in use, converts the fumaric acid
and ammonia to aspartic acid, which is industrially useful as sugar substitute (aspartame). The reaction is
shown below:

C4H4O4 + NH3 ßà C4H7O4N

Michaelis-Menten kinetics can be used for a good approximation of the reaction kinetics with Km = 4.0 g L-1.
The solution containing the substrate, ammonium fumarate, is in 15% (w/v). That is, 15.0 g substrate per 100
mL solution. The enzyme is added and the batch reactor is run until 85% of substrate is converted into
product. The effect of temperature is given as a priori and shown in the table below:

Temperature [oC] vmax [g L-1 h-1] Enzyme half-life [days]

32 5.90 10.50
37 8.50 2.30

a)Estimate the batch reaction time for both temperatures. Which operating temperature would you
recommend?
b)If the dumping, cleaning, and assembling time, which accounts for the downtime, is approximately 28 hours
per batch, at the temperature chosen in (a), what reactor volume is needed to produce 4000 tonnes of
aspartic acid per year?
 ILLUSTRATIVE EXAMPLE

In a 1,550-L batch reactor, an enzymatic reaction is used to produce an industrially useful

product. The kinetic parameters for the specific enzyme in use are as follows: vmax = 0.91 g L-1 h-
1; K = 1.5 g L-1. The starting substrate concentration for a single batch is 2.9 g L-1. Formation of
m
the product formed is stoichiometrically related to the amount of the substrate consumed with
a mass ratio of 1.20 : 1.00 (product : substrate). Operation cost, which includes labour,
maintainance, and energy and utilities, is of course proportional to the time that the reactor
operates and is estimated to be P216,000 per day. After the reaction, the resulting reaction
mixture contains the product, residual substrate, and the enzyme. Thus downstream processing
is needed to purify the mixture. Product recovery cost depends on two factors: (1) substrate
conversion (2) product concentration on the resulting reaction mixture. This can be
approximated using the equation below:

(for conversions between 70% and 100%)

where: C = cost in PhP (Philippine peso) per kilogram of product treated

X = substrate conversion in percentage
 ILLUSTRATIVE EXAMPLE
Assume a negligible product loss during purification. The product can be sold in the market
having a price of P33,750 per kg product. If the current operation is set for 75% substrate
conversion per batch,

a)Estimate the company’s net income from selling the product, that is excluding all the costs, in
PhP/batch.
b)Your boss proposed to operate the bioreactor until 90% substrate conversion is achieved,
validate the effect of this. Will the net income per batch will increase or decrease? By how
much?
c)As a newly-hired bioprocess engineer, you want to maximize the net income as to impress
your superiors. What substrate conversion should you propose?
 REFERENCES

Dr. David Shonnard, Chemical Engineering, Michigan Technological

University

Principles of Biochemistry, Lehninger, 4th edition

Bioprocess Engineering, Shuler, Kargi

Dr. Loh Kai Chee, Chemical and Biomolecular Engineering