Biochemistry Lecture No. 8: Enzyme Kinetics
Friday September 21 , 2012
Lysozymes As Catalytic Enzymes:
-A small, protective enzyme found in tears, saliva, mucus, and eggs, lysozymes hydrolyze
polysaccharides found in cell walls of some bacteria, which essentially breaks down their bacterial wall.
They perform cleavage on the sugars of the cell wall. A part of the innate immune system, it is stabilized
by disulphide bonds.
-The structure of the lysozyme contains two acidic residues (which are critical in allowing the enzymatic
reaction to occur) and a small cleft for an active site.
The Lysozyme Hydrolysis Mechanism:
-A chain of five sugar molecules (A, B, C, D, and E) is the substrate, which the lysozyme will act upon,
forming the enzyme substrate complex (ES complex). As this substrate approaches the lysozyme, it will
often cause its active site or cleft to change slightly. In between sugars D and E, the lysozyme forces
sugar D into a strained chair conformation. This makes it amendable for the next step of the reaction to
-The donation of a proton (H ) from glutamic acid (at position 35) to the oxygen holding the two sugars
together (D and E) transforms it into a hydroxyl group. This cleaves the bond between sugars D and E
The aspartic acid (at position 52), acting as a nucleophile, donates a pair of electrons to the hydrogen
(electrophile) in order to form a bond between oxygen and carbon 1 of sugar D. The substrate is now
covalently bonded to the lysozyme at the aspartic acid at position 52.
-In the last step, water donates a proton to both the carbon 1 of sugar D and the glutamic acid at
position 35. This action restores the lysozyme to its original state and completes the catalytic reaction.
Summary Of The Lysozyme Hydrolysis Mechanism:
Substrate distortion – change in sugar’s chair conformation.
Proton donors/acceptors – in strategic positions.
Transient covalent complex
Transition state stabilization – Decreases activation energy needed.
-Enzyme kinetics is defined as the study of how the rate of reaction is influenced by substrate
concentration, inhibitors, etc. and can help us discover how an enzyme works in a quantitative manner.
The rate of reaction will depend on various factors such as: the affinity of the enzyme for the substrate
and substrate’s concentration. - The rate (V) of an enzymatic reaction increases as the S concentration increases until a maximum rate
(V ) is reached. At V , all substrate-binding sites on the enzyme are fully occupied, and the rate of
the reaction is limited by the rate of the catalytic reaction on the enzyme’s surface (The nature of the
enzyme is the only limit