BIO3110 – BIOCHEMISTRY II-
INTERMEDIARY
METABOLISM
ENZYME AND ENZYME KINETICS
Catalysts of life
• Virtually all chemical reactions necessary for
maintaining life processes are mediated by
enzymes.
• Enzymes are composed of proteins except
for RNA modifying catalysts called
ribozymes.
• Ribozymes are RNA that catalyze reactions
on the phosphodiester bonds of other RNAs.
• Enzymes selectively channel reactants
(called substrates) into useful pathways.
Catalysts of life
• Enzymes determine whether a reaction can
take place or will take place.
• Many thermodynamically feasible reactions
in a cell that could occur do not proceed at
any appreciable rate (on their own).
• Biological catalysts speed up rates of
reactions that would otherwise be too slow to
support life.
• Enzymes direct all metabolic events.
What are enzymes?
• Enzymes are biological catalysts that speed
up chemical reactions in living organisms.
• There are more than five hundred different
enzymes in every cell of the body.
• Some enzymes work outside the cells, for
example the enzymes in the digestive
system.
• Enzymes ensure that the reactions happen
in the right place and at the right time.
Enzymes are Proteins
• Comprise of chains of amino acids
• A protein with catalytic properties due to its power of
specific activation
Nomenclature
• Each enzyme is assigned two names:
1. Short, recommended name
2. More complete systematic name
• Most enzyme names have the suffix “-ase” attached to the
substrate of the reaction (for example, glucosidase and
urease)
• Or to a description of the action performed (for example,
lactate dehydrogenase and adenylyl cyclase).
• While, some enzymes retain their original trivial names,
which give no hint of the associated enzymic reaction, for
example, trypsin and pepsin.
Systemic Names
• For a given enzyme, the suffix -ase is
attached to a fairly complete description of
the chemical reaction catalyzed, including
the names of all the substrates, for example,
lactate:NAD+ oxidoreductase.
• Each enzyme is also assigned a
classification number. Lactate:NAD+
oxidoreductase, for example, is 1.1.1.27.]
• The systematic names are unambiguous and
informative but are frequently too
cumbersome to be of general use.
Properties of Enzymes
Active site
• Enzyme molecules contain a special pocket
or cleft called the active site.
• The active site, formed by folding of the
protein, contains amino acid side chains that
participate in substrate binding and catalysis.
• The substrate binds the enzyme, forming an
enzyme–substrate (ES) complex.
• Binding is thought to cause a conformational
change in the enzyme (induced fit model)
that allows catalysis.
Properties of Enzymes
Active site
• ES is converted to an enzyme–product (EP)
complex that subsequently dissociates to
enzyme and product.
• Substrate binding depend on amino acids at
various positions.
Properties of Enzymes
Active site
• Of the 20 amino acids, only a few a involved
in the active site: cysteine, histidine, serine,
aspartate, glutamate and lysine
• These can participate in binding the substrate
and several serve as proton donors or
acceptors.
Properties of Enzymes
• Catalytic efficiency
• Enzyme-catalyzed reactions are highly
efficient, proceeding from 103–108 times
faster than uncatalyzed reactions.
• The number of molecules of substrate
converted to product per enzyme molecule
per second is called the turnover number, or
kcat, and typically is 102–104s-1.
Properties of Enzymes
Specificity
• Enzymes are highly specific, interacting with
one or a few substrates and catalyzing only
one type of chemical reaction. The set of
enzymes made in a cell determines which
reactions occur in that cell.
Properties of Enzymes
• Holoenzymes, apoenzymes, cofactors,
and coenzymes
• Some enzymes require molecules other than
proteins for enzymic activity.
• The term holoenzyme refers to the active
enzyme with its nonprotein component.
• Whereas the enzyme without its nonprotein
moiety is termed an apoenzyme and is
inactive.
Properties of Enzymes
• Holoenzymes, apoenzymes, cofactors,
and coenzymes
• If the nonprotein moiety is a metal ion, such
as Zn2+ or Fe2+, it is called a cofactor.
• If it is a small organic molecule, it is termed a
coenzyme.
• Coenzymes that only transiently associate
with the enzyme are called cosubstrates.
Cosubstrates dissociate from the enzyme in
an altered state (NAD+).
Properties of Enzymes
• Holoenzymes, apoenzymes, cofactors,
and coenzymes
• If the coenzyme is permanently associated
with the enzyme and returned to its original
form, it is called a prosthetic group (FAD).
Coenzymes commonly are derived from
vitamins. For example, NAD+ contains niacin,
and FAD contains riboflavin.
Properties of Enzymes
Regulation
•Enzyme activity can be regulated, that is,
increased or decreased, so that the rate of
product formation responds to cellular need.
1.Regulation of gene expression controls the
quantity and rate of enzyme synthesis.
2.Proteolytic enzyme activity determines the
rate of enzyme degradation.
3.Covalent modification of preexisting inactive
proenzymes produces active enzymes.
Properties of Enzymes
• Location within the cell
• Many enzymes are localized in
specific organelles within the cell.
• Such compartmentalization
serves to isolate the reaction
substrate or product from other
competing reactions.
• This provides a favorable
environment for the reaction and
organizes the thousands of
enzymes present in the cell into
purposeful pathways.
Chemical Reactions
• Before a chemical reaction can occur, the
activation energy barrier must be
overcome. There must be an input of energy.
• Molecules that could react with one another
often do not because the lack sufficient
energy.
• Each reaction has a specific activation
energy.
• EA is the minimum amount of energy
required before collisions between the
reactants will give rise to products.
Transition state
• Reactants need to reach an intermediate
stage called the transition state.
• The transition state has a higher free energy
than that of the initial reactants.
Transition State
MAKING REACTIONS GO FASTER:
• Increasing the temperature make molecules
move faster
• Biological systems are very sensitive to
temperature changes.
• Enzymes can increase the rate of reactions
without increasing the temperature.
• They do this by lowering the activation
energy.
• They create a new reaction pathway “a
short cut”
Activation Energy
• The only molecules that are able to react at a
given time are those with enough energy to
exceed the activation energy barrier.
• For most reactions at normal cell
temperature, the activation energy is so high
that few molecules exceed the EA barrier
• The EA barrier must be overcome in order for
the needed reactions to occur.
• This can be achieved by either increasing
the energy content of the molecules or by
lowering the EA requirement.
Activation Energy
• A catalyst enhances the rate of a reaction by
providing such a surface and effectively
lowering the activation energy.
• Catalysts themselves are unaltered by the
reaction, nor are they permanently changed
or consumed.
Catalytic Activation
MECHANISM OF ENZYME ACTIVITY
• The active site is the region of the enzyme
that binds the substrate by multiple weak
interactions to form an enzyme–substrate
complex, and transforms it into product.
• Two models have been proposed to explain
how an enzyme binds its substrate:
• the lock-and-key model and the induced-fit
model
MECHANISM OF ENZYME ACTIVITY
The Lock and Key Model of Enzyme
Activity
MECHANISM OF ENZYME ACTIVITY
The Induced Fit Model of Enzyme
Activity
• Some proteins can change their shape or
conformation
• When a substrate combines with an enzyme, it
induces a change in the enzyme conformation.
• The active site is then molded into a precise
conformation making the chemical environment
suitable for the reaction.
• The bonds of the substrate are stretched to
make the reaction easier (lowers the activation
energy).
• This explains the enzyme that can react with a
range of substrates of similar types.
Enzyme Assays
• An enzyme assay measures the conversion of
substrate to product, under conditions of cofactors,
pH and temperature at which the enzyme is optimally
active.
• High substrate concentrations are used so that the
initial reaction rate is proportional to the enzyme
concentration.
Enzyme Assays
• Either the rate of appearance of product or the rate of
disappearance of substrate is measured, often by
following the change in absorbance using a
spectrophotometer.
• E.g. Reduced nicotinamide adenine dinucleotide
(NADH) and reduced nicotinamide adenine
dinucleotide phosphate (NADPH), which absorb light
at 340 nm, are often used to monitor the progress of
an enzyme reaction.
Linked enzyme Assays
• If neither the substrates nor products of an
enzyme-catalyzed reaction absorb light at an
appropriate wavelength, the enzyme can be
assayed by linking it to another enzyme-
catalyzed reaction that does involve a
change in absorbance.
• The second enzyme must be in excess, so
that the rate limiting step in the linked assay
is the action of the first enzyme.
Isoenzymes
• Isoenzymes are different forms of an enzyme
which catalyze the same reaction, but which
exhibit different physical or kinetic properties.
• The isoenzymes of lactate dehydrogenase
(LDH) can be separated electrophoretically
and can be used clinically to diagnose a
myocardial infarction.
Allosteric Enzymes
• There is a class of enzymes, allosteric
enzymes, that bind small, physiologically
important molecules (allosteric effectors) to
modulate enzyme activity.
Classification
Factors affecting Enzymes
• substrate concentration
• pH
• temperature
• inhibitors
Substrate concentration:
Enzymic reactions
Vmax
Reaction
velocity
Substrate concentration
Faster reaction but it reaches a saturation point when all the enzyme molecules are
occupied.
If you alter the concentration of the enzyme then Vmax will change too.
Effect of pH
• Extreme pH levels will produce denaturation
• The structure of the enzyme is changed
• The active site is distorted and the substrate
molecules will no longer fit in it
• At pH values slightly different from the
enzyme’s optimum value, small changes in
the charges of the enzyme and it’s substrate
molecules will occur.
• This change in ionisation will affect the
binding of the substrate with the active site.
Michaelis Menten Equation
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