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The Beginning of Biochemistry
T Ramasarma
T Ramasarma is a INSA
Honorary Scientist at
SSCU and Department of
Biochemistry, Indian
Institute of Science,
Bangalore. He is a senior
biochemist and has
worked on biological
oxidation of free radicals.
Keywords
Fermentation, enzymes, catalysis, proteins, biochemistry.
1 This
article is based on a talk
given at the Zonal Workshop
on 'Emergence of Modern Sciences (1895-1905)', organized
in August 2000 by Mahesh
Chandra, Karnataka Rajya
Vijnana Parishat, at the Indian
Institute of Science, Bangalore.
Modern science often seems like a mighty river in flood showing
its awesome power, and reminds me of a Burman song on Ganga
- 'whither you come, whither you go? Take the River Kaveri:
from its origin in a small pond in a temple in Tala Kaveri in
Western Ghats, it flows on and is joined by many tributaries to
finally make the larger river on its way to the sea'. Many such
small beginnings took place during the decade of 1895-1905 and
later expanded into new fields of science. Biochemistry is one
such field that had its origin in that golden decade. A simple
observation of gas bubbles, indicating fermentation in a cell-free
yeast extract with added syrup, heralded the birth ofbiochemistry.
Fermentation by Living Yeast
From antiquity, humans have used microbes for producing
wine and vinegar, and for making bread, buttermilk and cheese.
Fermented foods, such as idli and dosa in southern India, are
nutritious and also popular all over the world. Although humans
all over the world knew how to make fermented foods, they did
not understand what was happening. Mysteries unknown are
often ascribed to divinity or devil. A great scientific advance of
the 19th century was the recognition that microbes cause fermentation. This realization shifted the emphasis to trying to
identify the particular microbes responsible for specific fermented foods or drinks. The identification of yeast as the microbe responsible for fermentation of grape juice to wine, and
molasses to beer, was one of the earliest great discoveries in
biotechnology. Since the time of Louis Pasteur (1822-1875), it
was known that during the growth of yeast, intact living cells
fermented sugars producing alcohol as a by product. At that
time it was considered impossible to have a life process such as
fermentation without living cells.
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Buchner's Classic Experiment
By the 19th century, the brewery industry had expanded into a
fine art, and a profitable one. Two German brothers, Hans and
Eduard Buchner, wanted to study the toxicity of yeast extract in
rabbits. This was a puzzling idea, as yeast extract was not known
to do any harm and, in fact, it is used as a vitamin supplement.
However, with this strange idea, the Buchner brothers went on
to do a remarkable experiment. First, they ground the yeast cells
with sand and then squeezed the mixture through a filter under
high pressure to obtain a clear extract, which they named 'zymase'. Incidentally, the procedure ensured that the extract was
free of intact cells, although it is not known whether this was
intended or not. With some unexplained logic, sugar syrup was
added to this extract hoping to protect it from other microbes
around. Lo and behold! Bubbles of gas came out, and fermentation to alcohol was seen for the first time to occur in extracts free
of cells (see Box 1). Thus did modern biochemistry begin. The
results of this and other experiments were recorded in a series of
papers starting in 1897 by one of the brothers, Eduard Buchner,
in the then most prestigious chemistry journal Berichte.
Enzymes in Yeast Extract, Zymase
Since the catalytic components responsible for fermentation
Box 1. Demonstration of Fermentation by Yeast
Active dry baker's yeast is now available in the market in the form of granules (Rs 10-15 for 25 g packet).
A teaspoon of these granules can be put in about 25 mllukewarm water for about 10 minutes until they
soften and become a fine suspension. Add half a teaspoon of cane sugar crystals (sucrose), or any sweet
syrup, to the yeast suspension and keep the mixture in warm water. In a few minutes copious bubbles of
carbon dioxide are produced. The reaction can be done in a transparent glass or plastic bottle for easy
visualization.
It is more difficult to do the Buchner experiment because yeast cells are hard to break. By grinding with
fine sand for several minutes with a mortar and pestle, a good fraction of cells break and release their
cellular contents and the enzymes. The clear extract after filtration can be used as above to show
fermentation. Remember Buchner used a high pressure filter press and ensured that intact cells did not
escape.
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were seen to be present in (en) yeast (zyme), W Kuhne derived
the apt name, 'enzyme' for the cellular catalyst. Basically the
fermentation of sugars to alcohol involves breakdown of 6carbon glucose to 2-carbon ethanol with optimum yield of about
30% by weight. Early experiments, mostly by German scientists,
focused on the way this breakdown of glucose occurred. How
was the glucose molecule processed? What were the intermediates? How many steps and enzymes were involved? These were
some of the first biochemical investigations. It was finally established that the process involved ten steps, each with a distinct,
specific enzyme designed to prepare the molecule from structural and energetic viewpoints. This reaction sequence, called
glycolytic pathway, emerged as the foundation of understanding chemical reactivity in living cells, referred to as metabolism.
Cell-free extracts became excellent source material for studying
many cellular enzymes. Biochemists revel in fractionation of
complex material and isolate the component responsible for an
activity under test. Thus began a primary biochemical expedition of 'enzyme hunt' and isolation of pure enzymes on a large
scale. Several enzymes, characterized by the reactions they
catalyse, were discovered. In most cases the name of the enzyme
includes the substrate and the reaction type, invariably ending
in 'ase'. The discoverers of zymase as the active yeast extract
were honoured by the adoption of this nomenclature.
Since the catalytic
components
responsible for
fermentation were
What are these Mysterious Enzymes?
seen to be present in
The investigators in this initial period of biochemistry were all
converts from chemistry who embarked on fractionating the
crude tissue extracts to isolate the enzymes in pure state and
characterize them. It soon became obvious that the living cell is
a bag full of enzymes endowed with a wide range of catalytic
potential. These enzymes were normally heat-sensitive, and
easily inactivated by chemical insults, particularly on treatment
with acids, alkalis and protein-damaging agents. Many enzymes
in crude cell-free extracts were isolated in pure state and snme in
crystalline form by following protein purification procedures.
W Kuhne derived the
(en) yeast (zyme),
apt name, 'enzyme'
for the cellular
catalyst. It soon
became obvious that
the living cell is a
bag full of enzymes
endowed with a wide
range of catalytic
potential.
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GENERAL
Enzymes give rate
4
enhancements of 10
to 10 14 , far beyond
the scope of nonprotein catalysts.
Comparing enzymes
and other catalysts is
like comparing the
relative speed of a
satellite and that of
an ant.
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However, the notion that enzymes were proteins was not widely
accepted.
R Willstater, an authority in those days, questioned the need for
a big, bulky protein for catalysis when it could be accomplished
in a laboratory by small molecular weight organic or inorganic
complexes. At best, it was thought, the protein may act as a
carrier, with an active site stuck on its surface, a notion perfectly
justified from the viewpoint of chemistry. But at that time, it
was not yet appreciated that enzymes give rate enhancements of
104 to 1014, far beyond the scope of non-protein catalysts. Comparing enzymes and other catalysts is like comparing the relative speed of a satellite and that of an ant. Moreover catalysis in
living cells must occur in water medium at low ambient temperature and neutral pH. For example, a solution of starch is
stable at room temperature for days, but in presence of tiny
amounts of saliva containing amylase it is hydrolysed in minutes (see Box 2).
Sumner's Stamina
In 1926 J B Sumner of Cornell University, New York (USA)
isolated a protein from jackbean that catalyzed the hydrolysis of
urea to ammonia and CO 2. This protein required just 1.4 sec to
Box 2. Demonstration of Activity of an Enzyme
Human saliva is a ready source of highly active a-amylase that degrades starch rapidly . This can be used
to demonstrate rapid action of an enzyme. Make a highly dilute solution of starch by adding a small amount
of starch powder (or rice powder) to boiling water (-0.1 gin 100 ml). The dilution can further be adjusted
such that the solution gives a blue colour with 1-2 drops of iodine reagent (60 mg iodine crystals and 30
g potassium iodine in 100 ml water; alternatively diluted tincture iodine can be tried) . The blue colour is
due to iodine fitting inside the helical structure of the polymeric starch molecule . The colour is lost when
the starch is hydrolysed to small units of glucose and maltose. A civilized way of obtaining saliva, other
than spitting, is to put a small strip of blotting paper (-0.5 cm x 5.0 cm) in the mouth for a few seconds
to wet it. Put this strip in the dilute starch solution for about 5 min with occasional shaking, and then add
the drops of iodine. No blue colour is formed when the starch is hydrolysed. A control strip dipped in water
can be tested in the same way . The time required depends on the amount of the enzyme and of starch. It
is important to use a very dilute solution of starch, just enough to obtain the blue colour with iodine.
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decompose its own weight of urea at 20° C. Fortunately for
Sumner and biochemistry, the protein crystallized on keeping a
31.6% acetone-water extract of jackbean powder in the cold.
Painstakingly, Sumner proved that the crystals were indeed
formed from the globular protein. He carefully stated in the
summary of his paper "I am compelled to believe that this
globulin is identical with the enzyme urease". Sumner then had
to face the might of the powerful German scientific establishment to prove that the whole protein he isolated was indeed the
active catalyst and not its mere carrier. This required a great deal
of confidence in his own experiments and convictions, and a lot
of stamina to withstand opposition and scrutiny from the scientific community. Sumner exhibited these qualities and established one of the most important concepts in biochemistry that
enzymes are indeed proteins.
Sumner established
one of the most
important concepts
in biochemistry that
enzymes are indeed
proteins. Since then,
the view that
enzymes are
proteins persisted
and became a
dogma.
There is an interesting human story behind this episode that 1
heard from Stewart of totipotency fame on a visit to our laboratory. It seems that Sumner had lost the palm of one of his hands
and, consequently, used to do his experiments by putting the
test tube in his pocket and then picking up the pipette to add the
reagents to the tube. Scientists in a German laboratory, being
initially unable to reproduce Sumner's experiment, resorted to
putting the tube in their pocket and working with one hand put
behind the back to jocularly reproduce the conditions of Sumner's
experiment.
Sumner later went to Germany and personally demonstrated the
experiment. Since then, the view that enzymes are proteins
persisted and became a dogma. It is only recently that catalytic
activity has been found in some form of nucleic acids, and these
are now called ribozymes. Thus, the homage to yeast (zyme)
continues.
The Present Profile
After a century of biochemical research, we know a great deal
about enzymes. The amino acid sequences and three-dimen-
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GENERAL
Address for Correspondence
T Ramasarma
INSA Honorary Scientist
Solid State and Structural
Chemistry Unit and Department of Biochemistry
Indian Institute of Science
Bangalore
560 012,
India.
I ARTICLE
sional structures are known for many enzymes and the active
site amino acids are also known. Large amounts of enzyme
proteins are now available, particularly by the use of cloning
techniques. They are being used as reagents the in laboratory,
and in industry and also as medicines. Yet we do not clearly
understand exactly what makes the big protein molecule work
so efficiently as a catalyst. Four principal structural features
form the core of the design ofa protein: peptide units, hydrogen
bonds, side-chains and globular fold. How does an enzyme use
these in its function? Often, it is thought that the site where the
substrate comes in contact with the protein is all we need to
know. What is the bulk of the protein doing besides providing
the tiny active site? How do they achieve the remarkable high
rates of catalysis in water and at ambient temperature? Amazing
versatility is achieved in making and breaking bonds by enzyme
proteins. Yet why does a protein depend on ribosomal-RNA, a
ribozyme, for synthesis of its own peptide units? The cellular
economy is better served by one protein having more than one
enzyme function. Each of the reactions catalyzed by a protein
may be specific but the protein has enough surface area to
provide more than one active site and thereby more activities. Is
this design more meaningful to the cellular activity beyond the
obvious, superficial advantage? Many more such questions remain to be asked and answered about enzymes.
~
'There are two ways to live in your life.
One is as though nothing is a miracle .
The other is as though everything is a miracle.'
- Albert Einstein
(1879-1955)
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