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TIP - Manila

363 P. Casal, Quiapo


City of Manila, Philippines
Website: www.tip.edu.ph
Tel. Nos. (+632) 733-9117 / (+632) 733-9142

CHEP 503: BIOCHEMICAL ENGINEERING

LECTURE 1:
ENZYME KINETICS
Lecturer:
ENGR. MICHAEL ALLAN G. RAMOS
Department of Chemical Engineering
Technological Institute of the Philippines
1st Semester, A.Y. 2016-2017

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.
- They dont change the equilibrium constant. not chemically altered
in reaction

ENZYMES
increase rate of reaction by providing a pathway of lower
activation energy to get from reactants to products
works by forming complexes with their substrates (binding), thus
providing unique microenvironment for reaction to proceed, the active
site. Interaction between enzyme-substrate complex is governed by
van der Waals forces and hydrogen bonding
- substrate binds to specific site on the enzyme known as the active
site
very high specificity/ selectivity for both reaction catalyzed and
substrate used. interaction is governed by lock-key concept.
very high catalytic efficiency

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, separated and
purified either intracellular or extracellular (separation process is
different) to obtain a formulated product to be used for an industrial
application
- 80% or their value is on industrial process biocatalysts

ENZYMATIC CATALYSIS

CO-FACTORS
- Requires amino acid residues for chemical activity
- Some requires cofactors to function (Fe2+ Mg2+ Mn2+ Zn2+) or
coenzymes one or more metal ions

CLASSIFICATIONS
- Naming: - ase
- Urea urease
- Polymerization of nucleotides to form DNA DNA polymerase
- Lyse bacterial cell wall Lysozome
- ATP: glucose phosphotransferase catalyzes transfer
of a phosphoryl group from ATP to glucose
-in, such as digestive enzymes, trypsin, chymotrypsin, pepsin
- depends on type of reaction
oxidaze - oxidation
hydrolase hydrolysis
dehydrogenase removal of hydrogen
- depends on subsrate
glucose oxidase
pyruvate carboxylase
succinate dehydrogenase

CLASSIFICATIONS

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

Batch reactor derivation and example

PLOTTING EXPERIMENTAL DATA

-good for low [S]


-for evaluating inhibitions

PLOTTING EXPERIMENTAL DATA

-Large errors but less bias for low [S]

-For accurate detm 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

ENZYME INHIBITION
- enzymes bind to certain compounds which reduces their activity
(recall CPI: how carbon deposits can reduce catalytic activity by occupying
active sites without prior hydrotreatment of feed gases to the FCCU)

- may be reversible and irreversible. Irreversible inhibitors; lead,


cadmium, mercury etc.)
simply treat with EDTA and citrate

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

v=

vm ,app .[S ]

k2

k m' ,app + [S ]
KI

Where;

vm ,app =

vm

[
I]
1 +

KI

km' ,app =

k m'

[
I]
1 +

KI

COMPLEX ENZYME KINETICS: INHIBITION


D) Substrate Inhibition

vm .[S ]

v=
k m'

2
[
S]
+ [S ] +

k2

S
S

KS

KS

[Smax]

S2
How to calculate for [Smax]?

dv
=0
d [S ]

[Smax ] =

km' .ks

SUMMARY OF INHIBITIONS

EFFECT OF TEMPERATURE

EFFECT OF pH

Optimal pH should be between pK1 pK2

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

km' ,app

[ ]

K2
H+
= km' .1 + + +

H
K
1

[ ]

REFERENCES
Dr. David Shonnard, Chemical Engineering, Michigan Technological
University
Bioprocess Engineering, Shuler, Kargi
Principles of Biochemistry, Lehninger, 4th edition
Dr. Loh Kai Chee, Chemical and Biomolecular Engineering

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