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Lecture

BIOL 200 Lecture Notes - Reaction Rate, Organic Chemistry, Daniel E. Koshland Jr.


Department
Biology (Sci)
Course Code
BIOL 200
Professor
Mathieu Roy

Page:
of 3
Enzymes are in general globular proteins and range from just 62 amino acid residues in size, for
the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid
synthase. A small number of RNA-based biological catalysts exist, with the most common being
the ribosome; these are referred to as either RNA-enzymes or ribozymes. The activities of
enzymes are determined by their three-dimensional structure. However, although structure does
determine function, predicting a novel enzyme's activity just from its structure is a very difficult
problem that has not yet been solved. Most enzymes are much larger than the substrates they act
on, and only a small portion of the enzyme (around 24 amino acids) is directly involved in
catalysis. The region that contains these catalytic residues, binds the substrate, and then carries
out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors,
which are needed for catalysis. Some enzymes also have binding sites for small molecules,
which are often direct or indirect products or substrates of the reaction catalyzed. This binding
can serve to increase or decrease the enzyme's activity, providing a means
for feedback regulation.Like all proteins, enzymes are long, linear chains of amino acids
that fold to produce a three-dimensional product. Each unique amino acid sequence produces a
specific structure, which has unique properties. Individual protein chains may sometimes group
together to form a protein complex. Most enzymes can be denaturedthat is, unfolded and
inactivatedby heating or chemical denaturants, which disrupt the three-dimensional
structure of the protein. Depending on the enzyme, denaturation may be reversible or
irreversible.Structures of enzymes with substrates or substrate analogs during a reaction may be
obtained using Time resolved crystallography methods.
Enzymes are usually very specific as to which reactions they catalyze and the substrates that are
involved in these reactions. Complementary shape, charge
and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this
specificity. Enzymes can also show impressive levels
of stereospecificity, regioselectivity and chemoselectivity. Some of the enzymes showing the
highest specificity and accuracy are involved in the copying and expression of the genome.
These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA
polymerase catalyzes a reaction in a first step and then checks that the product is correct in a
second step. This two-step process results in average error rates of less than 1 error in 100
million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are
also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes. Some enzymes that
produce secondary metabolites are described as promiscuous, as they can act on a relatively
broad range of different substrates. It has been suggested that this broad substrate specificity is
important for the evolution of new biosynthetic pathways.
Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil
Fischer in 1894 that this was because both the enzyme and the substrate possess specific
complementary geometric shapes that fit exactly into one another. This is often referred to as
"the lock and key" model. However, while this model explains enzyme specificity, it fails to
explain the stabilization of the transition state that enzymes achieve.
In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are
rather flexible structures, the active site is continuously reshaped by interactions with the
substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply
bind to a rigid active site; the amino acid side-chains that make up the active site are molded into
the precise positions that enable the enzyme to perform its catalytic function. In some cases, such
as glycosidases, the substrate molecule also changes shape slightly as it enters the active
site. The active site continues to change until the substrate is completely bound, at which point
the final shape and charge is determined. Induced fit may enhance the fidelity of molecular
recognition in the presence of competition and noise via the conformational
proofreading mechanism.
Lowering the activation energy by creating an environment in which the transition state is
stabilized (e.g. straining the shape of a substrateby binding the transition-state conformation of
the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition
state form, thereby reducing the amount of energy required to complete the transition).Lowering
the energy of the transition state, but without distorting the substrate, by creating an environment
with the opposite charge distribution to that of the transition state.Providing an alternative
pathway. For example, temporarily reacting with the substrate to form an intermediate ES
complex, which would be impossible in the absence of the enzyme.Reducing the reaction
entropy change by bringing substrates together in the correct orientation to react. Considering
ΔH alone overlooks this effect.Increases in temperatures speed up reactions. Thus, temperature
increases help the enzyme function and develop the end product even faster. However, if heated
too much, the enzyme’s shape deteriorates and the enzyme becomes denatured. Some enzymes
like thermolabile enzymes work best at low temperatures.
It is interesting that this entropic effect involves destabilization of the ground state and its
contribution to catalysis is relatively small.
The understanding of the origin of the reduction of ΔG requires one to find out how the
enzymes can stabilize its transition state more than the transition state of the uncatalyzed
reaction. It seems that the most effective way for reaching large stabilization is the use of
electrostatic effects, in particular, when having a relatively fixed polar environment that is
oriented toward the charge distribution of the transition state. Such an environment does not exist
in the uncatalyzed reaction in water.
The internal dynamics of enzymes has been suggested to be linked with their mechanism of
catalysis Internal dynamics are the movement of parts of the enzyme's structure, such as
individual amino acid residues, a group of amino acids, or even an entire protein domain. These
movements occur at various time-scales ranging from femtoseconds to seconds. Networks of
protein residues throughout an enzyme's structure can contribute to catalysis through dynamic
motions. This is simply seen in the kinetic scheme of the combined process, enzymatic activity
and dynamics; this scheme can have several independent Michaelis-Menten-like reaction
pathways that are connected through fluctuation rates. Protein motions are vital to many
enzymes, but whether small and fast vibrations, or larger and slower conformational movements
are more important depends on the type of reaction involved. However, although these
movements are important in binding and releasing substrates and products, it is not clear if
protein movements help to accelerate the chemical steps in enzymatic reactions. These new
insights also have implications in understanding allosteric effects and developing new medicines.
Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The
sites form weak, noncovalent bonds with these molecules, causing a change in the conformation
of the enzyme. This change in conformation translates to the active site, which then affects the
reaction rate of the enzyme. Allosteric interactions can both inhibit and activate enzymes and are
a common way that enzymes are controlled in the body.