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The important
ideas in science need time to gain acceptance.
For example, since the late nineteen fifties
that the precise images of crystallography have revealed
details of the interactions between
macromolecules and their molecular targets. It is easy,
then, to conclude that the recognition of the importance
of such interactions is a recent idea. Yet, in 1894,
twenty years before the structure of sodium chloride was
solved and more than half a century before any protein
structures were known, Emil Fischer, a
Dutch organic chemist, was able to propose that enzyme
and substrate fit together 'like a lock and key'.
Fischer's bold proposal was made after careful study of
the effects of enzymes on sugars and explained the
precise specificity of enzymes able to distinguish
between even the mirror images of the same molecular
structure.
Fischer's hypothesis withstood scientific
scrutiny, inspired Linus Pauling in his pioneering
model building as molecular biology began, and is now a
central theme in the rational design of drugs. As we
examine the precise registry between molecular entities
through computer graphics images in this section we
should not lose sight of the fact that the closeness of
the interactions involved was first understood more than
one century ago. We will see the ways in
which molecular recognition, as the specific intimate
interaction of molecules with one another is known,
influences all forms of microscopic chemical
communication.
Molecules have distinctive shapes and
correspondingly distinct properties. The realization
that chemicals were three dimensional entities came about
in the nineteenth century through the clear reasoning of
physical chemists like Le Bel and van't Hoff. Indeed, van
t'Hoff's successful models, proved to be the inspiration
for Fischer's explanation of an enzyme's ability to
differentiate, at the most subtle level, between
molecular shapes, yielding the original lock and key
hypothesis.
Although this framework for understanding enzyme activity
has now been in place for one hundred years it has taken
time, many experiments and analysis to add detail to the
intuitive understanding of the pioneering biochemists.
Naturally, there are
good reasons for the gradual adoption of three
dimensional chemical reasoning. The most important is
that although special properties could be explained
on the basis of unique shape - direct evidence of
the shapes involved was difficult to obtain.
The small size of molecules renders the
determination of structures difficult.
Indeed, molecular structure is still often difficult to
determine in many circumstances and in the past
this limitation has been even more pervasive than it is
today. The advantages that three dimensional structural
information bestow have become progressively more
apparent as molecular biology and structural chemistry
have advanced adding impetus to the drive to determine
yet more stuctures. An interesting example of the
delightful insights of stuctural information is provided
by the history of the study of the enzyme lysozyme. This
example again highlights the opportunities afforded by
the interaction between differing fields of scientific
research. Medicine, chemistry and crystallography
collided to provide the first glimpse of Fischer's
molecular 'lock'.
The medical need for improved treatments for bacterial
infection began the chain of events. Throughout the
majority of our history bacterial diseases have been
lethal to humans. Today, however, such infections can be
routinely treated with antibiotics. Our modern
exploitation of antibiotics began in 1929 when Dr.
Alexander Fleming, working at St. Mary's Hospital
Paddington, discovered penicillin, a compound excreted by
the penicillium strain of mould accidently growing on a
bacterial culture.The search for antibiotic compounds
was, however, a long one, and its discoveries were to
influence enzyme science in addition to therapeutic
medicine. In 1922 Dr. Fleming discovered that lysozyme,
an enzyme constituent of nasal mucus, was active against
bacterial cultures. Although Lysozyme, named because of
its ability to dissolve or lyse bacteria, had pronounced
anti-bacterial qualities, it was not effective against
the most lethal forms of bacteria. Lysozyme, however, was
found in, and excreted by, many living tissues, including
the tears of volunteer donors. Eventually a particulary
convenient source of the enzyme was found in the white of hens eggs.
Unlike many protein molecules, Lysozyme also proved to be
comparatively straightforward to extract, purify and
crystallize and for this reason proved to be an excellent
candidate for the emerging science of macromolecular
crystallography. Importantly, too, it was possible to
prepare crystals with differing heavy atoms incorporated
into the structure in the same position, making it
possible to reconstitute from diffracted X-ray
reflections accurate maps of the electron density of the
atoms of the lysozyme molecule.
In 1962 Lysozyme, then, became the first enzyme structure
to be determined. The lysozyme molecule is large in
comparison with many of the molecules you will see on
this site, containing around 1950 atoms. The
determination of their electron density map from hundreds
of diffracted X-ray intensities was an astonishing feat.
The work was carried out at the Royal Institution of
Great Britain by David Phillips and coworkers. The Royal
Institution has seen many scientific break throughs. It
was at the Royal Institution that Davy and Faraday
pioneered modern chemical and physical research and both
William and Lawrence Bragg, the discoverers and pioneers
of X-ray structural anlysis, were Laboratory Directors.
The backbond of the lysozyme protein, the active site is in the cleft at the center of the molecule
The images on the left are of a single molecule of the
enzyme lysozyme. The molecules contains 1950 atoms. The
upper image shows all the non-hydrogen atoms of lysozyme - this
image shows the molecular shape - but it is hard to see
the overall architecture of the molecule.The image to the
left conveys the shape of the lysozyme molecule with a
solid ribbon following the polypeptide backbone. Below we
show an image where the alpha helical portions of
lysozymes are highlighted with red cylinders.
This initial structural discovery was followed by the structural
complexes between the enzyme and bound saccharide
substrate. Lysozyme catalyzes the destruction of
bacterial cell walls through cleaving a particular link
between two sugar molecules. Understanding this reaction
illustrates the accuracy of Fischer's lock and key
hypothesis and also shows how the enzyme is able to bias
the natural reactive tendancy of a given molecule or set
of molecules to achieve a particular effect. The enzyme
is a natural catalyst. Just as the catalytic converter of
a modern car increases the rate at which the undesirable
products of internal cumbustion are combined with oxygen
and hydrogen to yield less harmful chemicals, so the
enzyme increases the rate at which a given chemical
transformation is achieved within a living organism. In
the case of lysozyme the reaction catalyzed is the
hydrolysis, or break down by adding water, of the linkage
between two sugar in a chain of sugar molecules. This
reaction might also be catalyzed using a strong acid.
Indeed, this is precisely the way in which starch may be
converted into useful sugar molecules industrially. But
concentrated acids are corrosive and difficult to handle
even in the laboratory and within a living organism will
indiscriminately attack
the vital structures of the host organism, such as
nucleic acids and protein molecules. What a living
organism needs is a catalyst specifically tailored to fit
the desired target or 'substrate'. Molecules which
precisely fit the active site of the enzyme lysozyme find
themselves acted upon by a powerful acid catalyst.
Molecules which do not fit the lock are safe from
destruction. The enzyme may even 'squeeze' the saccharide
molecule, in a molecular sense, to promote the formation
of the desired breakdown products. Other molecules, DNA
or hormones for example, do not fit the lysozyme active
site and thereby avoid the acid mediated destruction
which befalls the bacterial cell wall.
The registry of the
lysozyme molecule and the bacterial saccharide molecule
is highlighted in the image on the left. This image also
reveals the residues, the specific amino acids,
responsible for lysozyme's activity. The first active
amino acid is an acidic group, aspartame the thirty fifth
amino acid of the lysozyme chain, coloured yellow in the
diagram. This amino acid is placed on one side of the
binding groove occupied by the saccharide chain in the
image on the left. Residue thirty five is surrounded by
unreactive amino acids leading researchers to believe
that at the normal operating conditions for the enzyme it
is this amino acid which supplies the acidity necessary
to break the linkage between two sugars molecules. On the
opposite side of the enzyme's cleft is a negatively amino
acid side chain (which is also highlighted in the
diagram). When the sugar is attacked by the acid it
becomes positively charged and this second side chain
stabilizes the resulting molecular entity until it can
rearrange to the breakdown products of the catalytic
reaction. It is the cunning molecular architecture of the
enzyme which stabilizes the transition from intact to
cleaved bacterial cell wall and promotes the destruction
of a foreign cell.
The careful experimental investigation of the catalytic
properties of the lysozyme molecule heralded unparalleled
rationalization of a range of catalyic mechanisms. For
the first time it was possible to understand the
molecular constraints and appropriately poised functional
groups used by nature to build and destroy molecules,
cells and organisms. Many other catalytic mechanisms have
now been exposed by careful experimental investigation
and crystallographic work at a similar level of atomic
detail. Fischer's lock and key hypothesis has been shown
to be extremely accurate. As we shall see, the key-like
interaction of a molecule with a receptor site on a
macromolecule has also turned out to be an extremely
valuable way to understand the properties of many
molecules even when no direct information exists about
the actual structure of that receptor.
The structures revealed by today's experimental
techniques are averages representing many molecules and
their positions over long periods of time. However, a
static view of the entities is an over simplification.
Molecules must diffuse to and from the active site, often
carried by a dynamic sea of water molecules. Careful
measurement of the rates of enzymatic reactions and
protein folding have long indicated the existence of
considerable flexibility in even fully formed enzymes
structures. Indeed, computer simulations also highlight
the flexibility of Fischer's molecular locks as they
envelope and appropriately embrace their molecular keys.
The image on the left shows superimposed frames from a
simulation of a substrate in the active site of lysozyme
over a period of just 10-12
seconds.
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