Enzymes

Enzymes are proteins that have a catalytic function

Recall that a catalyst increases the velocity of a chemical reaction
No effect on equilibrium, as they facilitate both the forward and the reverse reaction
Example: Sucrose metabolism
One mol of sucrose releases 5700 kJ of energy when reduced
Now let’s review the following chemical reaction:

Sucrose + Oxygen can be oxidized – or metabolized – into carbon dioxide and water
Sucrose is very stable – this reaction cannot happen spontaneously
A catalyst facilitates the reaction
When this occurs non-biologically (e.g. burning the sucrose) it is simple chemistry

Introduction to Glycolysis

Cells undertake similar processes to derive energy; the oxidation of an energy rich
molecule like
glucose (another sugar) occurs within our cells daily
This is the basic reaction:

In the simple reaction, one glucose molecule is broken down into two pyruvate
molecules with a
net gain of 2ATP (the energy carrying molecule of our cells)
In terms of biochemistry, this process actually takes 10 separate steps, some of which
consume
energy, others of which release energy
 Glycolysis -Process Overview
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Now let’s review the high level step by step process for Glycolysis. In the next
section we will be reviewing each of these steps involved in Glycolysis in detail.

Glycolysis -Step 1
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In this step Glucose is first phosphorylated by a hexokinase enzyme to produce

Glucose-6-
phosphate (G6P). Note that this reaction consumes an ATP and as such is energy
consuming.
By phosphorylating glucose into G6P, the cell keeps the internal concentration of
glucose low,
which maintains osmotic flow of glucose into the cell in a positive fashion

Glycolysis -Step 2
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In this step G6P is rearranged into fructose-6-phosphate (F6P) by glucose phosphate
isomerase (as depicted below)
This isomerization process helps maintain stability of the reaction process

Important Note:
Notice how all the enzyme names have a common format:
End with ase
Name tends to describe it’s function
E.g phosphorylase adds a phosphate
 Glycolysis - Step 3
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In another energy-consuming step, F6P is modified into Fructose 1,6 bisphosphate
This is a non-reversible reaction and helps drive the energetics by destabilizing
the molecule

Glycolysis - Step 4
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In the next step, the destabilizing of the molecule in the previous reaction allows the
hexose ring to
be split by aldolase into two triose sugars: dihydroxyacetone phosophate (DHAP) and
glyceraldehyde
3-phosphate (GADP).
 Glycolysis - Step 5
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To simplify downstream reactions, triosephosphate isomerase now converts DHAP

into GADP, which
proceeds through the rest of the reactions
The dynamics is important – as GADP decreases in concentration through the
subsequent stages of
glycolysis
This reaction drives more DHAP to GADP because of the concentration differences

Glycolysis - Step 6
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This begins the “payoff” phase, where energy is produced – the conversion of GADP
to
1,3 bisphosphoglycerate (1,3BPG) results in the net production of an Nicotinamide
Adenine
Dinucleotide Hydrogen (NADH)

Glycolysis -Step 7
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The 1,3 BPG is now converted to 3-phosphoglycerate (3PG), producing an ATP

This is the break-even stage, since there are two 1,3BPGs created from one glucose at
the cost of 2
ATP’s, and each 1,3 BPG has now yielded an ATP, for a net change of zero ATPs
 Glycolysis -Step 8
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In a simple isomerization step, 3PG is converted to 2-phosphoglycerate (2PG)

Glycolysis - Step 9
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In this step Enolase now converts 2PG to phosphoenolpyruvate (PEP) as shown
below.
 Glycolysis - Step 10
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Lastly, PEP is converted to pyruvate by pyruvate kinase, releasing an additional ATP

Energy Change Table

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Let’s review the Energy Change Table which depicts the free energy that is
released during each step of the process of glycolysis.
 Important Note:
The above Energy Change Table shows that for each molecule of glucose that
proceeds through
the entire process of glycolysis, the change in free energy is approximately 76 kJ!
This energy is released by the molecules, which is why the free energy has a negative
value – the
reaction does not consume any net energy but rather releases it
Fermentation
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Glycolysis is only one essential enzymatic process, and as seen it actually requires 10
unique
enzymes.
Another important biological process is fermentation.
Fermentation is a metabolic process that converts sugars to acids, gases, and alcohol.
Occurs in yeast and bacteria, and in oxygen-starved muscle cells, as in the case of
lactic acid
fermentation
Used more broadly to refer to the bulk growth of microorganisms on a growth
medium, often
with the goal of producing a specific chemical product
There are many different types of fermentation, all of which are enzymatic.
Ethanol Fermentation
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The reaction begins with sucrose (a dimer of glucose and fructose)

Sucrose is cleaved into glucose and fructose by the enzyme invertase
The glucose is broken down through glycolysis, as before, into pyruvate
BUT! The pyruvate is then further broken down by pyruvate carboxylase, then
alcohol dehydrogenase, yielding ethanol and CO2
Energetically, this results in the same energy yield as glycolysis, but has the
additional benefit of the regeneration of NAD+, which is required for glycolysis, as
seen previously. Now let's review the Ethanol Fermentation process as shown
below.

Pepsin
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 Pepsin is a protease – which as you might guess is an enzyme which can break
down proteins
Pepsin is the primary digestive enzyme in our stomachs
Cleaves peptide bonds primarily between hydrophobic and aromatic amino acids
Important function is collagen digestion
Collagen is the fibrous portion of meats
Pepsin is capable of autolysis – it can cleave itself!
Now let’s review the structure of Pepsin and the hydrolysis of the peptide bond.

Ribozymes
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 Ribozymes are “enzymes” – catalytic, biologically active – but comprised of an
RNA which has formed a secondary structure.
RNA can be both genetic material (like DNA) and a biological catalyst (like
protein enzymes)
 RNA molecules that are capable of catalyzing specific biochemical reactions,
similar to the action of protein enzymes
Various functions, but primarily involved in RNA/DNA transactions – include in
the ribosome (protein synthesis)
The existence of ribozymes supports the RNA World hypothesis, which suggests
that the first “living” molecule to evolve on the Earth was an RNA
Now let's review a schematic of the Ribozyme cleavage of the RNA.

Lock & Key Hypothesis

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We have only looked at a few functional enzymes out of the thousands that exist
All of them function using what we suspect is the “lock and key” system first
suggested by Emil
Fisher
The lock represents an enzyme and the key represents the substrate it acts upon
Both enzyme and substrate have fixed, complementary conformations that lead to a
fit
At the active sites, the enzyme has a specific geometric shape and orientation that a
complementary substrate fits into perfectly
A more modern understanding suggests that the induced fit model is more
appropriate – enzymes and substrates are flexible and constantly in
motion – vibrating – and their proximity can induce the correct fit, provided they
are a match. Now let’s review the schematics of the Lock and Key Analogy as
shown below.
 PART 2
Introduction
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While enzyme names tend to be named for the substrate and reaction, this can
become unwieldy
So enzymes are additionally classified by their function using what is called an EC
code
The Enzyme Commission (EC) is a body of experts affiliated with the International
Union for
Biochemistry and Molecular Biology (IUBMB)
An EC contains 4 codes separated by periods (e.g. 1.2.3.4) where the first number
designates the
class of reaction performed by the enzyme
Top Level EC Codes
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The following is the list of the top level EC Codes and their 6 classes of enzymes.
 EC Specifiers
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Each level of the EC code adds specificity:
EC 3.4 are hydrolases that act on peptide bonds
EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid from
a polypeptide
EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide
Important Note:
Each unique enzymatic function, therefore, has a unique EC code.
Structural Proteins
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In addition to catalytic proteins, there are proteins that serves a structural function
Some of these are involved inside the cell, e.g. cell skeleton proteins
Others exist outside the cell
Cell/tissue binding
Communications & Signaling
Extra-Cellular Matrix
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The Extra-Cellular Matrix, or ECM, is a network of proteins and carbohydrates that is

excreted by cells
ECM occupies the space between cells (e.g. the epithelial and endothelial cells in this
diagram
Consists of four major components
Proteoglycans
Collagen
Elastin
Multi-adhesive Proteins
Now let’s review a schematic of the Extracellular matrix.
 Proteoglycans
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Proteoglycans are simply heavily glycosylated proteins – essentially proteins with

carbohydrate (think sugars!) bound to them
Proteoglycans form a thick matrix of negatively charged carbohydrates bound to a
protein core and serve as a “filler”
The carbohydrate binds high quantities of water, making the ECM elastic and
pressure-resistant
 Collagen
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Collagen is a triple-helical coil of three separate protein chains, each
approximately 1050 amino acids in length, with a core of repeated GLY-X-
Pro/Hy-Pro residues, where X can be any amino acid except TRP.
Collagen is a strong, resilient, fibrous protein, which resists tension forces
Hard and insoluble, almost 1/3 of all the protein in the human body is collagen
There are 16 different isoforms, but 90% of collagen in humans is comprised of
isoforms I, II, and III, which form fibrils.
 Elastin
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Elastin, as you may expect, forms elastic fibers, allowing tissue to elastically give to
pulling forces
and return to their original shape once the forces relent
This is a key characteristic of arteries, which need to expand and contract with blood
flow pressure
changes caused by the heart’s normal pumping behavior.
750 amino acids in length, elastin is 5 times as stretchable as an equal sized rubber
band.
Elastin is comprised of alternating hydrophilic and hydrophobic regions, which form
fluctuating
beta structures
Elastin is secreted into the folds in the cell surface and their forms fibers via cross-
linking between
lysine residues
 Cell Skeleton Proteins
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Many structural proteins serve to provide structure to the internals of the cell:
Microfilaments
Microtubules
Intermediate Filaments
These stabilize the shape of the cell as well as link internal cell structures

Now let's review a schematic of the same below.

 Microfilaments
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Microfilaments are comprised of actin:
7-9 nm in diameter
Responsible for cytokinesis, muscular contraction, and cell shape
Free actin tends to have ATP bound to it, and can polymerise into long helical
filaments called F-actin
Actin filaments in the cell are cross-linked into a network throughout the cytoplasm
Actin polymerization is required for muscular action; toxins such
as phalloidin and latrunculin act by blocking this polymerization
Many other proteins involved in positioning and shape bind to actin,
e.g. filamin, ankyrin, and dystrophin
Dystrophin is the protein which, in mutant form, is responsible for the disease
muscular dystrophy

Actin Polymerization
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Actin is a highly abundant (10-100 micromolar on average),~42 kDa structural
protein found in all eukaryotic cells (except for nematode sperm)
With more than 95% conservation in the primary structure, actin is one of the most
highly-conserved proteins [1]
The globular form of actin, known as G-actin, forms the basic unit for actin
filaments
In many cases actin filaments may bundle together with other actin filaments, or,
together with their associated motor proteins (e.g. myosin superfamily) form an
elaborate network known as the actin cytoskeleton
This occurs primarily at or near the plasma membrane
Actin filaments are highly dynamic and their polymerization is usually correlated
to their disassembly. Generally, actin filament polymerization occurs over three
phases:
Nucleation phase
Elongation phase
Steady state phase

Alpha & Beta Tubulin

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 Microtubules are formed of heterodimers of alpha and beta tubulin
Alpha and beta tubulin always bind in the same orientation, so that the resulting
tubulins are
considered polar in nature
Microtubule formation begins at the Microtubule Organizing Centre, or MTOC
Most cells contain only ONE MTOC
Other proteins can be found bound to microtubules; these are commonly referred to
as
Microtubule Associated Proteins, or MAPs

Microtubulin & Microtubule Assesmbly

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Microtubules are comprised of alpha and beta tubulin
24 nm in diameter, up to several hundred micrometers in length!
Responsible for chromosomal orientation during mitosis, intracellular trafficking,
cell motility
(e.g. cilia/crawling), and the cell wall organization in plants and yeast
Energy dependent (requires GTP)
Alpha + beta dimerize
Heterodimers aggregate
 Intermediate Filaments (IF)
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Intermediate filaments are comprised of keratin OR vimentin
10 nm in diameter
Responsible for mechanical force stability, growth of nerve axons
Bind to transmembrane proteins
Help stabilize tissues
Now let's review the schematic of the Intermediate Filaments (IF).
Types of Intermediate Filaments
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Now let’s review the different types of Intermediate Filaments.

Structure of Intermediate Filaments(IF)

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 IF-proteins contain long helical segments, with more unordered heads and tails.
Two IF-proteins dimerse in parallel with their helical segments winding around
each other.(coiled-coil held together mostly by hydrophobic interactions). Two
suchdimers then polymerise into an antiparallel tetramer. These
form protofilaments; four of those form protofibrils and four of those intermediary
filaments. The head-parts of the protein stick out from the filaments like
the fibres from a lamp-brush, which can be seen in EM-pictures of the IF.
Important Note:
In IF tetramers both ends are identical; there are no + and – ends in tubulin or
actin.
Structure of Intermediate Filaments(IF)
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Because IF proteins are very cell-type specific, they can be used to identify the source
of metastatic tumours
During mitosis, IFs are depolymerized; they repolymerize after division is complete.
Several other proteins have been found to bind IF proteins; these are called
Intermediate Filament Associated Proteins, or IFAPs
Plectin: crosslinks IF to microtubules and the lamin network in the nculeus
Ankyrin: connects IF to plasma membrane proteins and microtubules
PART 3
Introduction
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Many diseases are caused by mutations in enzymes. These can be the result of an
error at any level of the process of making a protein, including:
DNA sequence mutation
Regulatory errors
RNA transcription errors
mRNA translation errors
Protein folding errors
Sequence Mutations
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From the previous course, you should be aware that each codon (set of 3 bases) in the
DNA
sequence for a protein coding gene corresponds to a specific amino acid
Changes in the sequence can result in a change to the amino acid coded for in that
position; even
worse, the resulting codon may code for a STOP signal
Alternately, the deletion or insertion of bases in the DNA can result
in frameshift mutations
Now let's review the Sequence Mutations table listed below that highlights the
sequence mutations
at the first, second and third positions.
 Regulatory & Transcription Errors
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A regulatory error can occur in several places
Transcription control: e.g. the “wrong” amount of transcript produced, e.g. not
enough, or too much
Translation control: a shorter polyA tail, for example, will cause the mRNA to
degrade faster
Transcriptional errors can be caused if the RNApolymerase “misreads” the DNA,
inserting an
incorrect base into the RNA
RNApol-II has an error checking function
Error rate is approximately 2 x 10-6, or 2 errors in every million bases
DNA polymerases adds nucleotides to the 3' end of a strand of DNA. If a mismatch is
accidentally incorporated, the polymerase is inhibited from further extension.
Proofreading
removes the mismatched nucleotide and extension continues as shown in the DNA
Polymerase chart below.
 Splicing & Translation Errors
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Splicing Errors:
Recall that some genes have alternative splicing
sites

Not all splice variants are appropriate in all tissues

Translation Errors:
Translation errors are even less common: approximately 1 x 10-5, or 1 in
100,000 amino acids

But – this error rate results in 1 in 100,000 amino acids being wrong
An error rate of 1 x 10-6 in transcription, (i.e. 1 in 1,000,000 amino acids), may result
in fewer amino acid changes, depending on where the errors occur!
Protein Folding Errors
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As previously discussed, folding is not simply a matter of finding the lowest free
energy
Chaperones and chaperonins can bind the nascent amino acid chain and modify
which
residues are available to bind
The nascent amino acid chain begins folding while it is still being synthesized – so
the parts of
the chain that have already been translated may bind to themselves, making those
regions
unavailable to as-yet untranslated regions
Chaperones/chaperonins can prevent this preliminary folding, or modify folding in
other
ways
Let’s review the following schematic related to Protein Folding.
 Protein Folding Errors (cont'd)
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There are diseases that result from protein misfolding:
Protein folding errors are almost always due to an underlying sequence error in the
protein,
or in the chaperone/chaperonins involved in folding
There are cases where there is no sequence error – the reason for misfolding is
less clearly understood in cases such asthese