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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
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
ENZYME KINETICS
ENZYME KINETICS
MICHAELIS-MENTEN KINETICS
MICHAELIS-MENTEN KINETICS
How does the substrate concentration affect the order of the reaction?
TURN-OVER NUMBER
k 2 = kcat
BATCH EXPERIMENT
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)
v=
vm ,app .[S ]
k2
k m' ,app + [S ]
KI
Where;
vm ,app =
vm
[
I]
1 +
KI
km' ,app =
k m'
[
I]
1 +
KI
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
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