Recombinant DNA II;
Enzymes I
Andy Howard
Introductory Biochemistry
19 October 2010
Biochem: Recombinant II, Enzymes I
10/19/2010
What we’ll discuss
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Recombinant II
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Protein-protein
interactions
Genomics
Proteomics
PCR
Mutagenesis
Gene Therapy
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Enzymes
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Classes
Enzyme kinetics
Michaelis-Menten
kinetics: overview
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Protein-protein interactions
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One of the key changes in biochemistry over
the last two decades is augmentation of the
traditional reductionist approach with a more
emergent approach, where interactions
among components take precedence over
the properties of individual components
Protein-protein interaction studies are the
key example of this less determinedly
reductionist approach
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Two-hybrid screens
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Use one protein as bait; screen many
candidate proteins to see which one
produces a productive interaction with
that one
Thousands of partnering relationships
have been discovered this way
Some of the results are clearly
biologically relevant; others less so
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2-hybrid
screen
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X is bait, fused
to DNA binding
domain of GAL4
Y is target, fused
to transcriptional
activator portion
of GAL4
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Reporter constructs:
How to study
regulation
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Put a regulatory sequence
GFP
into a plasmid upstream of
Aequorea victoria
a reporter gene whose product
27kDa monomer
is easy to measure and visualize
0.9Å resolution
Then as we vary conditions, we can see how
much of the reporter gets transcribed
Example: Green Fluorescent Protein, which can
be readily quantified based on fluorescent yield
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Genomics
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Application of these high-throughput
techniques to identification of genetic
makeup of entire organisms
First virus was completely sequenced
in the late 1970’s
First bacterium: Haemophilus
influenzae, 1995
Now > 50 organisms in every readily
available phylum
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What’s been
sequenced?
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Cf. Table 12.1
This list includes
only completed
eukaryotic
projects with size
> 20 MB
One might study
multiple
individuals within
a species
Species
Category
Plasmodium
falciparum
Protist
23 1998
Trypanosoma
cruzi
Protist
67 2005
Caenorhabdites Nematode
elegans
Size,MB Date
88 1998
Arabidopsis
thaliana
Plant
119 2000
Drosophila
melanogaster
Insect
180 2000
Oryza sativa
Plant
389 2002
Mus musculus
Mammal
2717 2005
Homo sapiens
Mammal
3038 1999
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How genomics works
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A researcher who wishes to draw general
conclusions about structure-function
relationships may want to learn the sequence
(“primary structure”) of many genes and nongenomic DNA in order to draw sweeping
conclusions or build a library of genetic
constructs, some of which he will understand
and others he won’t
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Complete sequencing
of a genome
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Fragment chromosomes
Shotgun sequencing of fragments
Reconstitution based on overlaps
Cross-checking to compensate for errors
Interpretation
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Human genome
project
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QuickTime™ and a
decompressor
are needed to see this picture.
Effort began in late 1980’s
to do complete sequencing
of the human genome
Methods development was
proceeding rapidly during
the period in question so it
“finished” well ahead of schedule in 1999
Partly federal, partly private
Related efforts in other countries
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Time
3 Jul
2000
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What’s the point?
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Better understanding of both coding and
non-coding regions of chromosomes
Identification of specific human genes
Medically significant results
Statistical results (x% are Zn fingers…)
Variability within Homo sapiens or some
other sequenced organism by comparing
complete sequences or ESTs between
individuals
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Proteomics
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Analysis of the resulting list of expressible
(not necessarily expressed!) proteins
Often focuses on changes in expression
that arise from changes in environmental
conditions or stresses
Often useful to analyze mRNAs along with
proteins
Mass spectrometry is a key tool in
proteomics
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How MS
works in
proteomics
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QuickTime™ and a
decompressor
are needed to see this picture.
Cartoon from
Science Creative
Quarterly at
U.British
Columbia, 2008
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Amplification
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Prokaryotic and eukaryotic cells can,
through mitosis, serve as factories to
make many copies (> 106 in some
cases) of a moderately complex
segment of DNA—provided that that
segment can be incorporated into a
chromosome or a plasmid
This is amplification
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Polymerase
chain reaction
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QuickTime™ and a
decompressor
are needed to see this picture.
This is a biochemical tool that enables
Kary
incorporation of desired genetic material into a
Mullis
cell’s reproductive cycle in order to amplify it
Start with denatured DNA containing a segment
of interest
Include two primers, one for each end of the
targeted sequence
The sequence of events is now well-defined after
three decades of refinement of the approach
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PCR: the procedure
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Heat to denature cellular dsDNA and
separate the strands
Add the primers (ssDNA) and
polymerase
Heat again, then cool enough for ligation
Continue cycling to get many cell
divisions ~ 106-fold amplification
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Cartoon
version
QuickTime™ and a
decompressor
are needed to see this picture.
Image courtesy nobelprize.org
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PCR in
practice
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RT-PCR
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Variant on ordinary
PCR: starting point is
an RNA probe that
can serve as a
template for DNA via
reverse transcriptase
Once cDNA copy is
available, normal
PCR dynamics apply
QuickTime™ and a
decompressor
are needed to see this picture.
Cartoon courtesy Cellular &
Molecular Biology group at
ncvs.org
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Mutagenesis
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Procedure through which mutations are
introduced into genomic DNA
May be used:
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To generate diversity
To probe the essentiality of specific genes
To examine particular segments of genes
To alter properties of DNA or its mRNA transcript
or a translated protein
To provide information and material for gene
therapy
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Random mutagenesis
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DNA (often locally ssDNA) is exposed to
mutagens in order to introduce random
mispairings or increase the rate of
mispairing during replication
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Can involve ionizing radiation
Can involve chemical mutagens:
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Error-prone PCR
Using “mutator strains”
Insertion mutagenesis
Ethyl methanesulfonate
Nitrous acid and other nitroso compounds
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Site-directed mutagenesis
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Specific loci in DNA targeted for alteration
Typically involves excision, addition of altered
bases, and religation
Can be accomplished even in eukaryotic cell
systems
Many biochemical systems can be
systematically probed this way:
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To find essential amino acids in expressible
proteins
To see which amino acids are important
structurally
To examine changes at RNA level
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How do we use these tools?
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Already discussed significance of
complete sequencing efforts
Generally: amplification and expression
give us access to and control of
biochemical systems that otherwise have
to be isolated in their original setting
These methods enable controlled
experiments on complex systems
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Gene therapy
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Cloned variant of deficient gene is inserted
into human cells
Can be done via viral or other vector carrying
an expression cassette
Maloney murine leukemia virus (MMLV, or
retroviral approach) works for cassettes up to
9kbp; depends on integrating the cassette
into the patient’s DNA
Adenovirus works up to 7.5 kb: never gets
incorporated into host, but simply replicates
along with host
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Retroviral
approach
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Adenoviral
approach
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iClicker quiz, question 1
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In a yeast 2-hybrid experiment, the bait is
fused to
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(a) The DNA-binding domain of GAL4
(b) The transcriptional activator domain of
GAL4
(c) Both of the above
(d) Neither of the above.
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iClicker quiz, question 2
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The human genome contains
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(a) 115 MBp
(b) 389 MBp
(c) 3038 MBp
(d) 5373 MBp
(e) None of the above
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Enzymes
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Okay. Having reminded you that not all
proteins are enzymes, we can now zero in
on enzymes.
Understanding a bit about enzymes makes
it possible for us to characterize the kinetics
of biochemical reactions and how they’re
controlled.
We need to classify them and get an idea
of how they affect the rates of reactions.
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Enzymes have 3 features
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Catalytic power (they lower G‡)
Specificity
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They prefer one substrate over others
Side reactions are minimized
Regulation
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Can be sped up or slowed down by
inhibitors and accelerators
Other control mechanisms exist
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IUBMB Major Enzyme Classes
EC # Class
Reactions
Sample
Comments
1
oxidoreductases
Oxidationreduction
LDH
NAD,FMN
2
transferases
Transfer
big group
AAT
Includes
kinases
3
hydrolases
Transfer of
H 2O
Pyrophos
hydrolase
Includes
proteases
4
Lyases
Addition
across =
Pyr decarboxylase
synthases
5
Isomerases
Unimolecular rxns
Alanine
racemase
Includes
mutases
6
Ligases
Joining 2
substrates
Gln
synthetase
Often need
ATP
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EC System
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4-component naming system,
sort of like an internet address
Pancreatic elastase:
Category 3: hydrolases
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Subcategory 3.4: hydrolases acting on
peptide bonds (peptidases)
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Sub-subcategory 3.4.21: Serine
endopeptidases
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Porcine
pancreatic
elastase
PDB 3EST
1.65 Å
26kDa
monomer
Sub-sub-subcategory 3.4.21.36:
Pancreatic elastase
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Category 1:
Oxidoreductases
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General reaction:
Aox + Bred  Ared + Box
One reactant often a cofactor
Cofactors may be organic (NAD or FAD)
or metal ions complexed to proteins
Typical reaction:
H-X-OH + NAD+  X=O + NADH + H+
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Category 2:
Transferases
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a-ketoglutarate
These catalyze transfers of
groups like phosphate or amines.
Example: L-alanine + aketoglutarate 
pyruvate + L-glutamate
Kinases are transferases:
they transfer a phosphate from ATP
to something else
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pyruvate
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O
Category 3:
hydrolases
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Water is acceptor of
transferred group
Ultrasimple: pyrophosphatase:
Pyrophosphate + H2O ->
2 Phosphate
Proteases,
many other sub-categories
O-
HO-P-O-P-OH
O-
O
Pyrophosphate
(dianionic form)
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C=C
Category 4:
Lyases
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Non-hydrolytic, nonoxidative elimination
(or addition) reactions
Addition across a double bond or reverse
Example: pyruvate carboxylase:
pyruvate + H+  acetaldehyde + CO2
More typical lyases add across C=C
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Category 5:
Isomerases
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Unimolecular interconversions
(glucose-6-P  fructose-6-P)
Reactions usually almost exactly isoergic
Subcategories:
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Racemases: alter stereospecificity such that
the product is the enantiomer of the substrate
Mutases: shift a single functional group from
one carbon to another (phosphoglucomutase)
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Category 6: Ligases
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Catalyze joining of 2 substrates,e.g.
L-glutamate + ATP + NH4+ 
L-glutamine + ADP + Pi
Require input of energy from XTP (X=A,G)
Usually called synthetases
(not synthases, which are lyases, category 4)
Typically the hydrolyzed phosphate is not
incorporated into the product; it gets left
behind as a free product
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Enzyme Kinetics
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Kinetics: study of reaction rates and the
ways that they depend on concentrations
of substrates, products, inhibitors,
catalysts, and other effectors.
Simple situation A B under influence of
a catalyst C, at time t=0, [A] = A0, [B] = 0:
then the rate or velocity of the reaction is
expressed as d[B]/dt.
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Order of a
reaction
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A reaction is said to be firstorder in a particular reactant if
its rate is proportional to the
concentration of that reactant.
A reaction is first-order overall
if its rate is proportional to the
concentration of only one
reactant.
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Chart courtesy
Purdue Univ.
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[B]
Kinetics, continued
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In most situations more product will be produced
per unit time if A0 is large than if it is small, and
in fact the rate will be linear with the
concentration at any given time:
d[B]/dt = v = k[A]
where v is the velocity of the reaction and k is a
constant known as the forward rate constant.
Here, since [A] has dimensions of concentration
and d[B]/dt has dimensions of concentration /
time, the dimensions of k will be those of inverse
time, e.g. sec-1.
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t
More complex cases
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More complicated than this if >1 reactant
involved or if a catalyst whose concentration
influences the production of species B is
present.
If >1 reactant required for making B, then
usually the reaction will be linear in the
concentration of the scarcest reactant and
nearly independent of the concentration of
the more plentiful reactants.
In fact, many enzymes operate by
converting a second-order reaction into a
pair of first-order reactions!
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Bimolecular reaction
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If in the reaction
A+DB
the initial concentrations of [A] and [D]
are comparable, then the reaction rate
will be linear in both [A] and [D]:
d[B]/dt = v = k[A][D] = k[A]1[D]1
i.e. the reaction is first-order in both A
and D, and it’s second-order overall
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Forward and backward
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Rate of reverse reaction may
not be the same as the rate at
which the forward reaction
occurs. If the forward reaction
rate of reaction 1 is designated
as k1, the backward rate
typically designated as k-1.
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Multi-step reactions
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In complex reactions, we may need to keep
track of rates in the forward and reverse
directions of multiple reactions. Thus in the
conversion A  B  C
we can write rate constants
k1, k-1, k2, and k-2
as the rate constants associated with
converting A to B, converting B to A,
converting B to C, and converting C to B.
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[ES]
Michaelis-Menten
kinetics
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t
A very common situation is one in which for
some portion of the time in which a reaction is
being monitored, the concentration of the
enzyme-substrate complex is nearly constant.
Thus in the general reaction
E + S  ES  E + P
where E is the enzyme, S is the substrate, ES is
the enzyme-substrate complex (or "enzymeintermediate complex"), and P is the product
We find that [ES] is nearly constant for a
considerable stretch of time.
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