Enzyme Kinetics

rmax - it is the maximum reaction rate wherein even if increased, the substrate
concentration will no longer be affected.

Km - it is the substrate concentration at half of rmax.

- Michaelis-Menten constant.
- describes the affinity of the substrate to the enzyme.

Relationships in the Substrate Concentration, Enzyme Concentration, and Reaction Rate

1. The reaction rate is proportional to the substrate concentration (that is, first-order reaction) when the substrate
concentration is in the low range.
2. The reaction rate does not depend on the substrate concentration when the substrate concentration is high since the
reaction rate changes gradually from first order to zero order as the substrate concentration is increased.
3. The maximum reaction rate rmax is proportional to the enzyme concentration within the range of the enzyme tested.

Michaelis - Menten Equation (����)(��)

rp =
�� + ��
Rate Determining Reaction /
ES=> P+E
Step
Lock and Key Theory - the main concept of this hypothesis is that there is a topographical, structural
compatibility between an enzyme and a substrate which optimally favors the
recognition of the substrate.

Assumptions on Reaction Rate Equation Derivation

1. The total enzyme concentration stays constant during the reaction.

Total Enzyme Concentration
Equation CEO = CES + CE

2. The amount of enzyme is very small compared to the amount of substrate. Therefore, the formation of
enzyme-substrate complex does not significantly deplete the substrate.
3. The product concentration is so low that product inhibition may be considered negligible.

Approaches in Deriving the Rate Equation

1. Michaelis - Menten - Fast Equilibrium Step Assumption

Approach - Henri and Michaelis and Menten
- it is assumed that the product-releasing step is much slower than the reversible
reaction and the slow step determines the rate, while the other is at equilibrium
- it is often employed in heterogeneous catalytic reactions in chemical kinetics.
- the enzyme-substrate complex formation step is much faster than the product
releasing step which involves chemical changes.

2. Briggs - Haldane Approach - Pseudosteady - State Hypothesis

- G.E. Briggs and J.B.S. Haldane
- the change of the intermediate concentration with respect to time is assume to
be negligible, that is dCES/dt = 0.
- it is often used in developing rate expressions in homogeneous catalytic
reactions.

3. Numerical Solution - these are solution of the simultaneous differential equations developed withut
simplification.
 Effect of pH on Enzyme Activity

pH of solution - strongly influences the rate of enzyme reaction both in vivo and in vitro.
- the optimum pH is different for each enzyme.

Bell-Shaped Curve - the shape of the graph that shows the typical relationship between the reaction
velocity and pH.

Reasons that the Rate of Enzyme Reaction is Influenced by pH

1. Enzyme is a protein which consists of amino acid residues.

2. The amino acid residues possess basic, neutral, or acid side groups which can be positively or negatively charged at
a given pH.
Example:
- Glutamic Acid is acidic at lower pH. As the pH is increased, glutamic acid is ionized.
*Note: COOH is the first to be removed.
- Lysine is basic in the range of higher pH value. As the pH is decreaased, lysine is ionized as pH value of lysine
is 10.0 t which half of the residues are ionized.

3. An enzyme is catalytically active when the amino acid residues at the active site each possess a particular charge.
Therefore, the fraction of the catalytically active enzyme depends on the pH.

*Note: Variations in the pH of the medium result in changes in the ionic form of the active site and changes in the
activity of enzyme and hence the reaction rate.

Effect of Temperature to Enzyme Activity

- The rate of enzyme-catalysed reactions increases with temperature up to a certain limit.

- Above a certain temprature, enzyme activity decreases with temperature because of enzyme denaturation.

Temperature Activation - the ascending part; the rate varies according to the Arrhenius equation in this
region.

Temperature Deactivation / - the descending part; the percent maximal activity drastically decreases when
Temperature Denaturation the a certain temperature is exceeded.

Effect of Shear to Enzyme Activity

Fluid Shear - the mechanical force that an enzyme solution which is generated either by
flowing fluid, the shaking of a vessel, or stirring with an agitator.

Charm and Wong (1970) - they showed that the enzymes catalase, rennet, and carboxypeptidase were
partially inactivated when subjected to shear in a coaxial cylinder viscometer.

Thomas and Dunhill (1979) - they studied the effect of shear on catalase and urease activities by using a
coaxial cylindrical viscometer that was sealed to prevent any air-liquid contact.
- they found that there was no significant loss of enzyme activity due to shear
force alone at shear rates up to 106 sec-1.

*Note: Cellulose deactivation due to the interfacial effect combined with the shear effect was found to be far more
severe and extensive than that due to the shear effect alone.
 How Enzymes Work

- Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme -
substrate complex.
- Enzymes do not the affect the free - energy change or the equilibrium constant.

Noncovalent Forces that are Involved in Enzyme Activity

a. Electrostatic Interactions - these include charge-charge, dipole-dipole, charged-induced dipole, and
dipole-induced dipole interactions.
- the magnitude of these forces depends on the distance between molecules, all
depend inversely on the dielectric constant of the solvent between the ions or
dipoles.

b. Van der Waals Forces - comprised of electron cloud repulsion and attractive dispersion forces (London
forces). Dispersion forces are not large, but in an enzyme, the sum of all such
forces between substrate and enzyme may be quite significant.

c. Hydrogen Bonds - it is important in biological systems and occur when two electronegative atoms
are bound to a common proton. Often oxygen is one of the atoms.

d. Hydrophobic Forces - these reflect the tendency of apolar molecules to partition from an aqueous
environment to a hydrophobic one. The driving force for such movement can be
though as a result of the entropy gain when water molecules, which must be
structured around an apolar molecule, are able to assume a more random
arrangement when the molecule is transferred.
- the magnitude of this force is found experimentally to depend on the surface
area of the molecule.

Proximity Effect - it is where enzymes can hold substrates such that reactive regions of substrates
are close to each other and to the enzyme’s active site.
- this is the simplest mechanism by which an enzyme may enhance the rate of a
reaction.

Intramolecular Catalysis - a type of catalysis where one group adjacent to a reacting group provides
catalytic assistance in the reaction.
Example: Hydrolysis of tetramethyl succinanilic acid

Covalent Catalysis - a type of catalysis where an enzyme can form a covalent bond with one or more
reactants and so alter the reaction path from that observed in the uncatalyzed
case.

Electrostatic Catalysis - a type of catalysis that is not generally important in homogeneous catalysis in
aqueous systems.
- in this catalysis, the aromatic and aliphatic amino acid residues present at the
active site act to reduce the dielectric constant and charged amino acid residues
act as fixed dipoles, thus stabilizing charge quite effectively.

Catalysis Involving Metal Ions - in this type of analysis, a metal ion is present at the active site, and this ion
plays an important role in stabilizing negative charges that are formed in
electrophilic catalysis.

Zinc, Copper and Cobalt - metalloenzymes that are commonly involved in coordination of oxyanions
involved as reaction intermediates.