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Enzymes
Enzymes: Biological catalysts that
promote and speed up chemical
reactions without themselves being
altered (consumed) in the process.
They determine the patterns of
transformations for chemicals, as
well as forms of energy in the living
organisms.
Historical Events in Discovery of
enzymes as biocatalysts-1
• Both enzymology and biochemistry were evolved from
the 19th century investigation on the nature of animal
digestion and fermentation.
• Biochemical reactions could not be reproduced in the
lab initially and was thought (e.g., Louis Pasteur) to
occur by the action of a “vital force”.
• The idea of “catalytic force” or “contact substance”
promoting fermentation was introduced in about 1830s.
• Addition of alcohol to an aqueous extract of malt
(geminating barley) and saliva precipitated a flocculent
material which liquefied starch paste and converted it
into sugar, this material was named diastase (1833)
(later amylase).
Enzymes as the biocatalysts-2
• Pepsin was discovered as the active principle in the acid
extract of gastric mucosa causing the dissolution of
coagulated egg white (1834).
• Other “soluble ferments” discovered in the 19th century
include trypsin (1857), invertin (later invertase and sucrase,
1864), papain (“vegetable trypsin”, 1879), etc.
• “Enzyme (something in yeast) ” was first coined for such
“unorganized ferments” by Kühne in 1876.
• Enzymes for alcoholic fermentation were found to be active
in cell free extracts of yeast (1897, Eduard Buchner):
fermentation is a chemical process, not a “vital process”.
Enzyme specificity was revealed
by studying sugar conversion
• Sugars of known structure were synthesized and
used as substrates of enzymes.
• The a-methylglucoside was found to be
hydrolyzed by invertin, but not by emulsin,
whereas the b-methylglucoside was cleaved by
emulsin, but not by invertin: the enzyme and the
glucoside was considered to fit (complement)
each other like a lock and a key.
Enzymes as the biocatalysts (3)
• Relationship of initial velocity (V0) and substrate
concentration (S) was examined.
• A mathematical description was established for
the kinetics of enzyme action (Michaelis and
Menten, 1913). Before it was known that enzymes are proteins!!!
• Weak-bonding interactions between the enzymes
and their substrates were proposed to distort the
substrate and catalyze a reaction (Haldane, 1930s).
Leonor
Michaelis
(1875-1949)
A German
Maud
Menten
(1879-1960)
A Canadian
John Burdon
Sanderson
Haldane
(1892-1964)
A British
Geneticist
Formation of an enzyme-substrate
(ES) complex was suggested
• The activity of invertase in the presence of sucrose
survives a temperature that completely destroys it if the
sucrose is not present (C O’Sullivan and F. W. Tompson,
1890).
• Emil Fisher’s study on the specifity of invertase (1894).
• The rate of fermentation of sucrose in the presence of
yeast seemed to be independent of the amount of sucrose
present, but on the amount of the enzyme (A. J. Brown,
1902).
• The kinetics of enzyme action was originally studied using
invertase (a hyperbola when V0 was plotted against [S]).
• The enzyme (E) was thus assumed to form a complex. (ES)
with the substrate (S) before the catalysis.
The kinetics of the enzyme-catalyzed
reaction were found to be rather
different from those of a typical
chemical reaction
The rate is proportional to the
concentration of the reactant
in a typical chemical reaction.
Enzymes however showed
a saturation kinetics:
formation of ES complex
was hypothesized (1902).
Enzymes were found to be
proteins
• The question of homogeneity of the enzyme preparations
frustrated the field of enzymology for many decades.
• Nitrogen content analysis and various color tests (for
proteins) led to contradictory results.
• Filterable coenzymes (co-ferments) were discovered in
Buchner’s zymase (Harden and Young, 1906).
• Enzymes were thought to be small reactive molecules
adsorbed on inactive colloidal material, including proteins
( as by R. Willstätter in the 1920s).
• Urease (1926, Sumner) and pepsin (1930, Northrop) were
crystallized and found to be solely made of proteins.
Urease crystals
Sumner, J. B. (1926) “ The
isolation and crystallization
of the enzyme urease” J.
Biol. Chem. 69:435-441.
Pepsin crystals
Northrop, J. H. (1930)
“Crystallin pepsin, 1:
Isolation and tests of
purity” J. Gen . Physiol.
13:739-766.
The Nobel Prize in Chemistry 1946
“ for his discovery "for their preparation of enzymes and
that enzymes
virus proteins in a pure form"
can be
crystallized"
James Batcheller
Sumner
John Howard Northrop
Wendell Meredith
Stanley
1/2 of the prize
1/4 of the prize
1/4 of the prize
Cornell University
Ithaca, NY, USA
Rockefeller Institute for
Medical Research
Princeton, NJ, USA
Rockefeller Institute for
Medical Research
Princeton, NJ, USA
1887-1955
1891-1987
1904-1971
Not all enzymes are proteins: Some RNA
molecules (ribozymes) were found to be catalytic
(Sidney Altman and Thomas Cech, 1989).
Ribozymes are found to promote RNA
processing.
Sidney Altman visiting PKU
The enzyme theory of life was
formulated
• Enzymes are central to every biochemical
process (Hofmeister, 1901): “life is short and
thus has to be catalyzed”.
• Isolation, purification and physico-chemical
characterization of enzymes would be
important for understanding the nature of life.
• Without catalysis, the chemical reactions
needed to sustain life could not occur on a
useful time scale.
• Self replication and catalysis are believed to be
the two fundamental conditions for life to be
evolved. (RNA is thus proposed to be the type
of life molecules first evolved).
Rate enhancement
Fe3+ →1000 fold
Hemoglobin → 1 ,000,000 fold
Catalase → 1 ,000,000,000 fold
2 H2O2 → 2 H2O + O2
200,000 catalytic events/second/subunit
(near the diffusion-controlled limit).
The reaction is sped up by a billion fold!
(a prosthetic group)
Active site
(tetramers)
The current understanding
on the general features of
enzymes
Extraordinarily powerful;
Highly specific;
Be often regulated.
Trival name
• Gives no idea of source, function or reaction
catalyzed by the enzyme.
• Example: trypsin, thrombin, pepsin.
Systematic Name
• According to the International union Of
Biochemistry an enzyme name has two parts:
-First part is the name of the substrates
for the enzyme.
-Second part is the type of reaction
catalyzed by the enzyme.This part ends with
the suffix “ase”.
Example: Lactate dehydrogenase
EC number
Enzymes are classified into six different groups
according to the reaction being catalyzed. The
nomenclature was determined by the Enzyme
Commission in 1961 (with the latest update
having occurred in 1992), hence all enzymes
are assigned an “EC” number. The
classification does not take into account
amino acid sequence (ie, homology), protein
structure, or chemical mechanism.
EC numbers
• EC numbers are four digits, for example a.b.c.d,
where “a” is the class, “b” is the subclass, “c” is
the sub-subclass, and “d” is the sub-sub-subclass.
The “b” and “c” digits describe the reaction, while
the “d” digit is used to distinguish between
different enzymes of the same function based on
the actual substrate in the reaction.
• Example: for Alcohol:NAD+oxidoreductase EC
number is 1.1.1.1
The Six Classes
•
•
•
•
•
•
EC 1. Oxidoreductases
EC 2. Transferases
EC 3. Hydrolases
EC 4. Lyases
EC 5. Isomerases
EC 6. Ligases
• Additional information on the sub-subclasses and sub-subsubclasses (i.e, full enzyme classification and names) can be found
at the referenced web link.
• From the Web version,
http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html
EC 1. Oxidoreductases
• EC 1. Oxidoreductases :catalyze the transfer
of hydrogen or oxygen atoms or electrons
from one substrate to another, also called
oxidases, dehydrogenases, or reductases.
Note that since these are ‘redox’ reactions,
an electron donor/acceptor is also required
to complete the reaction.
EC 2. Transferases
• EC 2. Transferases – catalyze group transfer
reactions, excluding oxidoreductases (which
transfer hydrogen or oxygen and are EC 1).
These are of the general form:
• A-X + B ↔ BX + A
EC 3. Hydrolases
• EC 3. Hydrolases – catalyze hydrolytic
reactions. Includes lipases, esterases,
nitrilases, peptidases/proteases. These are of
the general form:
• A-X + H2O ↔ X-OH + HA
EC 4. Lyases
• EC 4. Lyases – catalyze non-hydrolytic (covered
in EC 3) removal of functional groups from
substrates, often creating a double bond in the
product; or the reverse reaction, ie, addition of
function groups across a double bond.
• A-B → A=B + X-Y X Y
• Includes decarboxylases and aldolases in the
removal direction, and synthases in the addition
direction.
EC 5. Isomerases
• EC 5. Isomerases – catalyzes isomerization
reactions, including racemizations and cistran isomerizations.
EC 6. Ligases
• EC 6. Ligases -- catalyzes the synthesis of
various (mostly C-X) bonds, coupled with the
breakdown of energy-containing substrates,
usually ATP
Mechanism