Biology Lab 173 - Midterm

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Enzymes

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Biological catalysts which accelerate and steer chemical processes
Proteins
Lower the activation energy necessary for the reaction to proceed
By lowering the minimum energy needed, more reactant molecules will possess enough energy to react
Lower activation energy by forming temporary union between the enzyme and substrate

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Properties of Enzymes

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Determined by their structure
Consists of amino acids linked together and folded into a complex three-dimensional conformation

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Defining Characteristic of a Protein

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The conformation

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Active Site

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Some amino acids from the folded peptide chain make up the catalytic region of the enzyme
Wghere substrates bind to the enzyme and where the reaction takes place
Shape and chemical composition of the amino acids of the active site determine which reactions that enzyme can catalyze

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Cofactors

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metal ions or organic molecules
Must be present in addition to the enzyme and the substrate molecules for the reaction to proceed at a rapid rate
Usually bind to, and form part of the active site

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Catalysts

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participate in reaction but are not changes
Neither reactants nor products and are not consumed in the reaction

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Substrates

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the reactants of enzyme-catalyzed reactions

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Enzyme - Substrate Reaction

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occurs when enzyme combines with substrate
Once products form, complex dissociates
Low concentration of enzyme can catalyze a reaction at a rapid rate, since the enzyme is not consumed and the formation and breakdown of the enzyme-substrate complex is extremely rapid

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Enzyme Activity

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measured by determining the rate of the catalyzed reaction

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Rate of Reaction

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change in concentration of a reactant over a period of time
May be expressed either as substrate disappearance or as product formation

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Enzyme Activity is Affected By...

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Concentration of enzyme, substrate, product, and the conformation of the enzyme
Presence of inhibitors

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Rates of Reactions Increase with...

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increases in concentrations of enzymes and substrates - temperature and pH are critical too since they influence enzyme conformation

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Inhibitors

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chemicals that decrease the rate of an enzymatic reaction - quantified by the percent inhibition

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Percent Inhibition

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the percent drop in enzyme activity in the presence of the inhibitor

(rate without in) - (rate with in)
rate without inhibitor

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Competitive Inhibitors

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molecular structure resembles that of the substrate, so they are able to bind to the active site of the enzyme
Bind to the active site, the enzyme molecule is unavailable to substrate molecule
Do not permanently bind to the active site

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How To Tell If it is a Competitive Inhibitor

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At high substrate concentrations relative to the concentration of inhibitor, the rate of reaction will be higher (because it is more likely that the substrate will "win" the competition for the active site)
Percent inhibition will be lower

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Noncompetitive Inhibitors

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do not bind to the active site but they reduce the ability of the enzyme to process the substrate
Substrate binding is unaffected
May remove necessary cofactor, bind to enzyme and reduct flexibility, change shape of the enzyme

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How to Tell if it is a Noncompetitive Inhibitor

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Affected by enzyme concentration and inhibitor concentration, but not substrate concentration
Percent inhibition remains constant as substrate concentration is varied

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Polyphenoloxidase

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enzyme found in both plants and animals
Isolate it from potatoes
Produces quinones, which appear to be toxic to many microbes and insects
Responsible for the darkening that occurs when freshly peeled fruit or potatoes stand in fresh air
Reaction catalyzed by polyphenoloxidase is exemplified by oxidation of catechol

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Catechol

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substrate used in the experiment
Oxidation of catechol consumes oxygen and produces benzoquinone (reddish-brown color)
Concentration can be measured with a spectrophotometer to determine the rate

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Homogenate

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suspension of broken cells that contain the enzyme

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Homogenization

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process of preparing a homogenate
Take the source of enzyme (a potato) cut it into small pieces, and blend it in distilled water
Ruptures cell membranes releasing also enzyme cofactors, substrates, and products

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Centrifugation

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after the homogenization
Spin homogenate at high speed - heavy particle will move to the bottom of the centrifuge tube - supernatant will be collected (liquid that contains enzyme)

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Serial Dilution

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diluting each tube by half by adding the dilution from the source to the other

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Control: Amount of Enzyme

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The higher the concentration of enzyme, the faster the reaction will occur. With no enzyme, there is a reaction that occurs in nature, but it is very slow.

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Control: Amount of Substrate

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The higher the amount of substrate, the faster the reaction will occur. However, there is a limit because if there is too much substrate then the solution will be "saturated" and there will be a point where there is only so much enzyme to react with the substrate and the reaction rate will level off

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Control: Temperature

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Enzyme structure is crucial for its function - the structure is affected strongly by its conditions
Initial rates allow us to tell which reaction is most effective and the speed
The optimum temperature for polyphenoloxidase is 45 degree Celsius because the initial rate is fastest at this temperature at 0 degrees Celsius it is slowest and at 65 it is believed to be denaturing because the reaction is slower

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Control: Cofactors and Cheleating Agents

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Cheleating agents added were
PTU - removes copper
KCN - removes copper and iron
Both cheleating agents inhibited the enzyme reaction which tells us for sure that copper is necessary for the reaction - need another cheleating agent that checks just iron in order to determine if it is necessary as well

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Control: Increasing Salt

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As one adds more salt to a reaction, it begins to denature the reaction and it will be slower

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Control: Potassium Arsenite

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non-competitive inhibitor - the % inhibition of the substrate will be the same for all levels of potassium arsenite added
Does not bind to the active site but does things such as removing cofactors or changing the shape of the enzyme

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Control: BHPA

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competitive inhibitor - the % inhibition of the substrate will change
Binds to active site - resembles the substrate itself and blocks substrates

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Control: Temperature (Boil, Freeze)

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When you freeze and thaw the enzyme, it has a normal enzyme reaction, comparative to the control
When you boil the enzyme is denatures it and it will not react with the substrate

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Gene

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length of DNA that codes for a biologically useful product

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Plasmids

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double-stranded circular DNA molecule
Replicate independent of chromosomal control (allows for amplification)
Carry non-essential (but sometimes advantageous genes)

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pGLO - Plasmid Template

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carries three genes and an ori
araC
GFP
bla - beta lactamase

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GPF

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green fluorescent protein, the gene we are trying to clone

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araC

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a regulatory protein, links GFP expression to presence of arabinose

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bla

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an enzyme that breaks down ampicillin and makes plasmid-bearing cells antibiotic resistant

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ori

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origin of replication, a region of plasmid DNA where proteins bind to initiate DNA replication (needed to make more copies of the plasmid in cells)

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Regulation of araBAD genes in E.coli

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AraC regulates araBAD genes - needed to import and use arabinose
AraC can act as a repressor or as an activator - when arabinose binds to AraC it turns it into its activator form

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Regulation of GFP in pGLO

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in pGLO gene is downstream of an AraC regulated promoter
Thus, arabinose turns on GFP production

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PCR

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amplifies tiny amounts of source DNA by replication
Denaturation - heat pGLO - strands separate
Annealing: cool to allow primers to form hydrogen bonds
Extension: DNA polymerase (Taq polymerase) adds nucleotides to the 3' end of each primer
Repeat - 25-30 times resulting in a billion fold

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Aragose Gel Electrophoresis

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DNA is negatively charged thus in an electromagnetic field it will move towards the cathode
Smaller fragments move farther and faster in a given amount of time
DNA is stained with EtBr which makes it glow in UV light
Should be about 831 base pairs

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Restriction Enzymes

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xBaI - bind at DNA at a specific site and cleave phosphodiester bond

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DNA Ligase

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use enzyme to join two restriction fragments after the sticky ends combine

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Vectors

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A gene by itself will not be replicated
Must be attached to something with replication controls (ori)

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Column Chromatography

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method of separating and purifying proteins based on some property they possess
Hydrophobicity
GPF is hydrophobic, when we change the salt concentrations, it denatures GFP and the hydrophobic residues are exposed and will stick to the beads in the column
As salt concentration is lowered, return to normal folding and no longer stick to beads

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Transformation

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DNA is mixed with E.coli and heat shocked in order to transfer some of the DNA into the E.coli cells

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GFP Gene from PCR product and insert into cloning vector

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PCR Cleanup
Digestion
Heat-inactivate restriction enzyme
Mix and ligate PCR fragment with vector

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PCR Cleanup

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to get rid of excess primer and nucleotides
PBI buffer added - removes primers - flow through the column matrix during centrifugation
Wash with PE buffer - discard flow-through
Centrifuge to dry
Elution buffer - longer DNA fragments released from column
Centrifuge - extra product is the PCR product

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Digestion

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to make compatible ends on the PCR product and plasmid vector
PCR product and restriction enzyme
Plasmid vector and restriction enzyme - this ensures that they have compatible ends

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Heat Inactivation of XbaI

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to make sure ligation is not undone

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Ligation

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to join fragment and vector