Bio 173 Exam Review

What are the steps in the scientific method?

Explore/Observe, Ask a question, conduct research, form hypothesis, experiment, analyze data and conclusion (if hypothesis is supported report results)

Think: Eating Quinoa Really Hurts Ethan’s Awful Cousin

E- Explore/observe

Q- question

R - research

H- hypothesis

E- experiment

A - analyze data

C- conclusion

Molecular System of enzyme

enzyme + substrate ←> ES Complex ←> Enzyme + Product

Why do scientists use the scientific method?

to acquire reliable and objective knowledge with little bias, help to prove causation instead of simply correlation

Hypothesis

possible explanation that leads to a TESTABLE prediction and motivates experiments

Prediction

a description of an expected outcome in the test (experimental) group and in each control group during an experiment

When are predictions made in an experiment?

AFTER an experiment is designed

What is the difference between a hypothesis and a prediction?

A hypothesis explains the WHY of a phenomenon while a prediction forecasts the exact results or IF you will record during a test; a hypothesis can lead to many predictions

Positive vs. negative control

positive control answers “can this experiment detect the effect is it’s present'“, while negative control answers “is the observed result due to the test variable or contamination?”

Non-specific inhibition

Positive Control

known, measurable positive response/effect

What is the purpose of a positive control? Give an example

verifies the experiment is working correctly; ex. using a sample with a known virus in a COVID test

Negative Control

no specific experimental change/effect

What is the purpose of a negative control? Give an example

ensures there’s no effect where it should be; ex. using pure water instead of a patient sample

As product is formed, we would expect absorbance to _____

increase

What is an enzyme?

a protein (usually) that is a biological catalyst ie. speeds up reactions without being consumed

Independent vs dependent variables

Independent variable is manipulated and depend is the measured outcome

What does an enzyme do?

speeds up the rate by lowering the activation energy barrier of a reaction

What does polyphenoloxidase do?

non-essential enzyme that causes browning in fruits and vegetables

How do enzymes lower the activation energy barrier

it provides a favorable microenvironment by 1) stressing substrate bonds and 2) orienting substrates correctly

What is metabolism?

the totality of an organism’s chemical reactions

A metabolic pathway_____

begins with a specific molecule and ends with a produce

How does increasing the concentration of a substrate influence catalysis?

The rate generally stays the same (if the enzyme is fully saturated)

How does decreasing the concentration of a substrate influence catalysis?

rate decreases due to less enzyme-substrate interactions (or nothing if enzymes are saturated)

How does increasing the concentration of an enzyme influence catalysis?

rate increases because if more frequent collisions with substrate

How does decreasing the concentration of an enzyme influence catalysis”

rate decreases because there is less enzyme to interact with

What is enzyme saturation? How does changing it affect catalysis?

Enzyme saturation happens when all of its active sites are filled by substrate molecules; this keeps the reaction rate constant and unchanging

What is the effect of salt on an enzyme?

ions in salt solutions can destabilize ionic bonds that hold the enzyme in its proper conformation; it denatures the enzyme

Why do extreme heats make enzymes unworking?

• Causes irreversible denaturation

• High heat weakens the bonds holding the protein structure together

• Destroys the shape of the active site, stopping enzymatic activity

What do cold temperatures to do enzyme rates?

• Preserves protein structure

• Cooler temperatures slow kinetic activity

• denatures the protein but it’s renatured once thawed

How do we perform a serial dilution? For 1/2? 1/4? 1/8?

each new dilution uses the previous solution as the source; for ½ add 500 ml undiluted into 500 ml water, for ¼ take 500 from this mixture and add to 500 ml water, for 1/8 take 500 from the ¼ concentration and add to 500ml water

Why do we need to add the enzyme right before starting a reaction?

to prevent the reaction from occurring before it can be measured

What reaction does polyphenoloxidase catalyze?

catechol into orthoquinone; oxidizes polyphenolics in quinones

How did we measure enzyme function?

By measuring product formation over time; rate of reaction

Why does the control Enzyme-Only have a detectable absorbance reading, but Catechol-Only does not?

How do we measure reaction rate?

slope of the first two minutes of the absorbance v time reaction to account for errors

How does [substrate] affect the reaction rate?

How does [enzyme] affect the reaction rate”

Why do product vs time graphs level off?

The reaction has finished; the substrate has run out or the enzyme is fully saturated

Why do initial rate graphs level off?

Micropipetting techniques

Spectrophotometry techniques

select wavelength, calibrate using a blank

Serial dilution

a step-wise series of dilutions where the dilution factor stays the same

What is Vmax and how is it measured?

It is the maximum rate of a reaction; it is measured when 1) all enzyme active sites are full (saturated) and 2) determined after multiple readings

What is Km?

When the reaction is half maximal, _____

What does Km do?

Competitive Inhibitor

• binds to active site

• COMPETES with substrate

• slows down reaction

Non-competitive inhibitor

How can you design an experiment to tell if a substance is an inhibitor?

If there is an inhibitor in the experiment, how do you determine if it is a competitive or noncompetitive inhibitor?

How to calculate percent inhibition and what do the results mean?

Percent Inhibition = (rate without inhibitor - rate with inhibitor) / rate without inhibitor; the results measure how effectively an inhibitor slows enzymatic activity

What is percent inhibition measuring?

It is calculated by comparing the enzyme's activity with the inhibitor against its maximum possible activity

How is Vmax affected by competitive vs noncompetitive inhibitors

Competitive inhibitor: vmax is unchanged because the reaction dilutes and beats out competitors

Non-competitive inhibitor: since it completely changes the composition of the enzyme, vmax is stunted and is much lower

How is Km affected by competitive inhibitors?

What are the differences in competitive and non competitive inhibitor reactions on a Rate vs [Substrate] graph?

Cofactor

non-protein enzyme helpers such as metal ions (inorganic) or vitamins (organic)

What is the role of cofactors?

activates the enzyme

Chelator

“chemical claw” that pulls metal ions from solution and inhibit enzyme function

What is a chelator used to determine? How?

What is the biological role of an enzyme?

Gut microbiome

Where is the gut microbiome located in the human body?

Large intestine (colon)

Why is dietary fiber important to maintain a healthy gut microbiome?

Fiber is ________ by the gut microbiome.

Fermentation

The process of breaking down fiber and intermediate products to produce gases and energy

How is fiber fermented by the gut microbiome? What are the products?

Explain how products of fermentation in the gut can be detected in the breath?

Gases produced by microbes in the large intestine (H2 and CH4):

1) Gases produced during microbial fermentation of fiber in colon

2) Diffuses into blood stream

3) Released from blood into lungs

4) Exhaled in breath

Why is it important to collect breath samples at the end of our exhalte?

Why do some people produce more methane gas in their breath?

Spread Plating

Streak Plating

What is the goal of spread plating?

What is the goal of streak plating?

Isolate a species from a colony; first step in gut microbiome experiment

Streak plating vs spread plating

Collecting breath samples techniques

Analyzing breath samples techniques

Freezer stocks

Why are freezer stocks prepared?

What are the basics of a Polymerase Chain Raction

What are the three steps of each PCR cycle?

What is in a PCR cocktail (master mix)? What is the function of each?

What is the purpose of positive and negative controls in PCR?

Since enzymes _______ energy, they are _______

release, exergonic

Exergonic vs Endergonic

exergonic is release of free energy that would occur eventually and endergonic absorbs free energy and are non spontaneous

Explain competitive inhibitor enzyme kinetics

Explain non-competitive inhibitor enzyme kinetics

What does spectrophotometry measure?

how much light absorbs or transmits as a function of wavelength

What is the enzyme-substrate Complex?

when substrates are help in active site of enzyme by weak interactions

Draw graph and label the stages when an enzyme catalyzes it

As enzyme concentration increases, reaction rate _____

increases

As substrate concentration increases, reaction rate _____

stays the same

Write the catalytic cycle equation

1) Substrates enter active site

2) Substrates are held in active site by weak interaction

3) The active site lowers activation energy

4) substrate are converted into products

5) Products are released

6) Active site is availible for new substrates

How do you calibrate a spectrophotometer

What explains the “wobble” seen in absorbance vs time curves?

Why is it important to measure initial rates?

Why does the reaction slow towards the end of a catechol and enzyme curve?

Each enzyme has an _______ environment it functions it (favors the most active shape). When not _______, it ________.

optimal, optimal, denatures

Non-competitive inhibitor relationship to vmax:

Since non-com inhibitors completely change the composition of an enzyme, it stunts the vmax

Competitive inhibitor relationship to vmax: