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Lecture 9

Lecture 9.docx


Department
Biological Sciences
Course Code
BIOD60H3
Professor
Shelley A.Brunt
Lecture
9

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Lecture 9: Mechanisms of Enzyme Action Continued
Progress of the reaction: transition state and intermediates during catalysis
The progress of a reaction can be represented by an energy diagram, or energy profile.
Figure 6.1 (slide 2) is an example that shows the conversion of a substrate (reactant) to a
product in a single step. The y axis shows the free energies of the reacting species. The x
axis, called the reaction coordinate, measures the progress of the reaction, beginning
with the substrate on the left and proceeding to the product on the right. This axis is not
time but rather the progress of bond breaking and bond formation of a particular
molecule. The transition state occurs at the peak of the activation barrierthis is the
energy level that must be exceeded for the reaction to proceed. The lower the barrier the
more stable the transition state and the more often the reaction precede.
Catalysts create reaction pathways that have lower activation energies than those of
uncatalyzed reactions.
Catalysts participate directly in reactions by stabilizing the transition states along the
reaction pathways.
Enzymes are catalysts that accelerate reactions by lowering the overall activation energy.
Catalysts accelerate reactions by lowering the overall activation energy.
Energy diagram for a reaction with intermediates
Energy diagram for a reaction with an intermediate - The intermediate occurs in the
trough between the two transition states. The rate determining step in the forward
direction is formation of the first transition state, the step with the higher energy
transition state. S represents the substrate, and P represents the product.
The slowest step, the rate-determining or rate-limiting step, is the step with the highest
energy transition state.
Catalytic mechanisms
Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Proximity and orientation effects
Preferential binding of transition state
Chemical Modes of Enzymatic Catalysis
The formation of an ES complex places reactants in proximity to reactive amino acid
residues in the enzyme active site.
Ionizable side chains participate in two kinds of chemical catalysis; acidbase catalysis
and covalent catalysis.
A. Polar Amino Acid Residues in Active Sites

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The active site cavity of an enzyme is generally lined with hydrophobic amino acid
residues. However, a few polar, ionizable residues (and a few molecules of water) may
also be present in the active site.
Polar amino acid residues (or sometimes coenzymes) undergo chemical changes during
enzymatic catalysis. These residues make up much of the catalytic center of the enzyme.
Types of ionizable residues found in the active sites of enzymes
Table 6.1 lists the ionizable residues found in the active sites of enzymes. Histidine,
which has a pKa of about 6 to 7 in proteins, is often an acceptor or a donor of protons.
Aspartate, glutamate, and occasionally lysine can also participate in proton transfer.
Certain amino acids, such as serine and cysteine, are commonly involved in group-
transfer reactions. At neutral pH, aspartate and glutamate usually have negative charges,
and lysine and arginine have positive charges. These anions and cations can serve as sites
for electrostatic binding of oppositely charged groups on substrates.
pKa values of ionizable groups of amino acid residues in proteins can differ than in free
amino acids
The microenvironment can change the pKas within a protein
pH can affect the reaction rate
Only a small number of amino acid residues participate directly in catalyzing reactions.
Most residues contribute in an indirect way by helping to maintain the correct
three-dimensional structure of a protein.
Enzymes usually have between two and six key catalytic residues. The top ten
catalytic residues are listed in Table 6.3. The charged residues, His (Histidine),
Asp (Aspartic acid), Arg (Arginine), Glu (Glutamic acid), and Lys (Lysine)
account for almost two-thirds of all catalytic residues. This makes sense since
charged side chains are more likely to act as acids, bases, and nucleophiles. They
are also more likely to play a role in binding substrates or transition states. The
number one catalytic residue is histidine. Histidine is 6 times more likely to be
involved in catalysis than its abundance in proteins would suggest.
There are roles of amino acid residues not directly involved in catalysis is to assist or
enhance the catalytic residues
Catalytic residues in addition to being directly involved in catalysis can also be involved
in:
Substrate binding
Stabilization of transition state
Interaction with cofactors
B. AcidBase Catalysis
In acidbase catalysis, the acceleration of a reaction is achieved by catalytic transfer of a
proton.
Acid–base catalysis is the most common form of catalysis in organic chemistry and it’s
also common in enzymatic reactions.

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Enzymes that employ acidbase catalysis rely on amino acid side chains that can donate
and accept protons under the nearly neutral pH conditions of cells.
This type of acidbase catalysis, involving proton-transferring agents, is termed general
acidbase catalysis. (Catalysis by H+ or OH- is termed specific acid or specific base
catalysis.)
In effect, the active sites of these enzymes provide the biological equivalent of a solution
of acid or base. It is convenient to use B: to represent a base, or proton acceptor, and BH+
to represent its conjugate acid, a proton donor. (This acidbase pair can also be written as
HA/A- .)
A proton acceptor can assist reactions in two ways: (1) it can cleave OH, NH, or even
some CH bonds by removing a proton
And (2) the general base B: can participate in the cleavage of other bonds involving
carbon, such as a CN bond, by generating the equivalent of OH- in neutral solution
through removal of a proton from a molecule of water.
C. Covalent Catalysis
In covalent catalysis, a substrate is bound covalently to the enzyme to form a reactive
intermediate. The reacting side chain of the enzyme can be either a nucleophile or an
electrophile. Nucleophilic catalysis is more common. In the second step of the reaction, a
portion of the substrate is transferred from the intermediate to a second substrate. For
example, the group X can be transferred from molecule AX to molecule B in the
following two steps via the covalent ES complex XE:
Concept: the ability to couple reactions is an important property of an enzyme
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