recombinant dna ii; enzymes i

10/19/2010Biochem: Recombinant II, Enzymes I

Recombinant DNA II; Enzymes I

Andy HowardIntroductory Biochemistry

19 October 2010

10/19/2010 Biochem: Recombinant II, Enzymes I

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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 sequenceinto a plasmid upstream ofa reporter gene whose productis 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

GFPAequorea victoria

27kDa monomer0.9Å

resolution


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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’sto do complete sequencingof the human genome

Methods development wasproceeding rapidly duringthe period in question so it“finished” well ahead of schedule in 1999

Partly federal, partly private Related efforts in other countries

QuickTime™ and a decompressor

are needed to see this picture.

Time3 Jul 2000


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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




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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



Kary Mulli

s


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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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Cartoon version



Image courtesy nobelprize.org


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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



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 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.

Chart courtesyPurdue Univ.


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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-2as 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

recombinant dna ii; enzymes i

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enzymes igenomicsapplication

enzymes iwhats

enzymes icomplete sequencing

enzymes ireporter constructs

enzymes itwohybrid screensuse

enzymes i2hybrid screenx

green fluorescent protein