10/22/2009biochem: recombinant ii, enzymes i recombinant dna ii; enzymes i andy howard introductory...
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10/22/2009Biochem: Recombinant II, Enzymes I
Recombinant DNA II; Enzymes I
Andy HowardIntroductory Biochemistry
22 October 2008
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What we’ll discuss
Recombinant II: Protein-protein interactions
Genomics Proteomics PCR Mutagenesis Gene Therapy
Enzymes Classes Enzyme kinetics Michaelis-Menten kinetics: overview
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Protein-protein interactions
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
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
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 Put a regulatory sequence into a plasmid upstream of a reporter gene whose product is easy to measure and visualize
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 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? 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
Size,MB
Date
Plasmodiumfalciparum
Protist 23 1998
Trypanosomacruzi
Protist 67 2005
Caenorhabditeselegans
Nematode
88 1998
Arabidopsisthaliana
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 A researcher who wishes to draw general conclusions about structure-function relationships may want to learn the sequence (“primary structure”) of many genes and non-genomic 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
Fragment chromosomes Shotgun sequencing of fragments Reconstitution based on overlaps Cross-checking to compensate for errors
Interpretation
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Human genome project
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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What’s the point?
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 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
Cartoon from Science Creative Quarterly at U.British Columbia, 2008
QuickTime™ and a decompressor
are needed to see this picture.
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Amplification 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
This is a biochemical tool that enables incorporation of desired genetic material into a 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 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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PCR in practice
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RT-PCR 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 Procedure through which mutations are introduced into genomic DNA
May be used: 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 DNA (often locally ssDNA) is exposed to mutagens in order to introduce random mispairings or increase the rate of mispairing during replication Can involve ionizing radiation Can involve chemical mutagens:
Error-prone PCR Using “mutator strains” Insertion mutagenesis Ethyl methanesulfonate Nitrous acid and other nitroso compounds
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Site-directed mutagenesis 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: 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?
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 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 In a yeast 2-hybrid experiment, the bait is fused to (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 The human genome contains
(a) 115 MBp (b) 389 MBp (c) 3038 MBp (d) 5373 MBp (e) None of the above
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Enzymes 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 Catalytic power (they lower
G‡) Specificity
They prefer one substrate over others
Side reactions are minimized Regulation
Can be sped up or slowed down by inhibitors and accelerators
Other control mechanisms exist
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IUBMB Major Enzyme ClassesEC
#Class Reaction
sSample Comments
1 oxidoreductases
Oxidation-reduction
LDH NAD,FMN
2 transferases Transfer big group
AAT Includes kinases
3 hydrolases Transfer of H2O
Pyrophos hydrolase
Includes proteases
4 Lyases Addition across =
Pyr decar-boxylase
synthases
5 Isomerases Unimolec-ular rxns
Alanineracemase
Includesmutases
6 Ligases Joining 2 substrates
Gln synthetase
Often need ATP
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EC System 4-component naming system,sort of like an internet address
Pancreatic elastase: Category 3: hydrolases
Subcategory 3.4: hydrolases acting on peptide bonds (peptidases)
Sub-subcategory 3.4.21: Serine endopeptidases
Sub-sub-subcategory 3.4.21.36: Pancreatic elastase
Porcine pancreat
ic elastasePDB 3EST
1.65 Å26kDa
monomer
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Category 1:Oxidoreductases General reaction:Aox + Bred Ared + Box
One reactant often a cofactor (see ch.7)
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
These catalyze transfers ofgroups like phosphate or amines.
Example: L-alanine + -ketoglutarate pyruvate + L-glutamate
Kinases are transferases:they transfer a phosphate from ATP to something else
-keto-glutarate
pyruvate
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Category 3:hydrolases
Water is acceptor of transferred group
Ultrasimple: pyrophosphatase:Pyrophosphate + H2O ->2 Phosphate
Proteases,many other sub-categories
HO-P-O-P-OH
O
OO-
O-
Pyrophosphate(dianionic form)
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Category 4:Lyases
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
C=C
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Category 5: Isomerases
Unimolecular interconversions(glucose-6-P fructose-6-P)
Reactions usually almost exactly isoergic
Subcategories: 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 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 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
A reaction is said to be first-order 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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Kinetics, continued 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.
[B]
t
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More complex cases
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 If in the reactionA + D Bthe 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 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 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 Cwe can write rate constantsk1, 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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Michaelis-Menten kinetics
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 "enzyme-intermediate complex"), and P is the product
We find that [ES] is nearly constant for a considerable stretch of time.
[ES]
t