173 Exam 1

Hypothesis vs. Prediction

• A hypothesis is a testable statement about the relationship between variables.
• A prediction is a specific outcome expected if the hypothesis is true.

Positive vs. Negative Control

• A positive control group is expected to produce a desired effect, confirming the experiment is working correctly.
• A negative control group is expected to show no effect, serving as a baseline for comparison.

What is an Enzyme?

• Enzymes are biological catalysts, speeding up chemical reactions without being consumed.
• All enzymes are catalysts, but not all catalysts are enzymes.

How Enzymes Lower Activation Energy

• Enzymes lower activation energy by:
  • Binding to reactants (substrates) and stabilizing the transition state.
  • Orienting substrates correctly.
  • Providing a favorable environment for the catalytic cycle.

Factors Affecting Enzyme Catalysis

• Find time: Time for an enzyme to find the next substrate.
• Processing time: Time for the enzyme to convert substrates into products.
• Enzyme concentration: Higher enzyme concentration increases reaction rate, but eventually levels off due to substrate limitation.
• Substrate concentration: Higher substrate concentration increases reaction rate, but eventually levels off due to enzyme saturation.
• Temperature and pH: Optimal temperature and pH maximize enzyme activity.
• Efficiency of enzyme: Different enzymes have different catalytic efficiencies.
• Factors affecting enzyme shape: Factors like temperature, pH, and inhibitors can alter enzyme shape and affect activity.

Vmax and Km

• Vmax: The maximum rate of reaction when all active sites are saturated.
• Km: The substrate concentration at which the reaction rate is half maximal.
• Higher Vmax and lower Km indicate a better enzyme, meaning less substrate is needed to reach half Vmax.

Role of Cofactors

• Cofactors are non-protein molecules or ions that assist enzymes in catalysis.
• They are essential for many enzymes to achieve full activity.

Competitive vs. Noncompetitive Inhibitors

• Competitive inhibitors bind to the active site, competing with substrates. They don't change Vmax but increase Km (lower enzyme affinity).
• Noncompetitive inhibitors bind to a different site on the enzyme, altering its shape and reducing Vmax. Km remains unchanged.

Identifying Competitive/Noncompetitive Inhibition

• Analyze the effect of inhibitors on Vmax.
• If Vmax is lowered, it's a noncompetitive inhibitor.
• If Vmax remains the same, it's a competitive inhibitor.

Percent Inhibition

• Competitive inhibitors show varying percent inhibition at different substrate concentrations.
• Noncompetitive inhibitors have constant percent inhibition across substrate concentrations.

Science Communication

• Science communication aims to increase public awareness, enjoyment, interest, opinions, and understanding of science.
• Barriers include scientific literacy, cultural differences, misinformation, and lack of trust in scientific sources.

Polyphenol Oxidase Lab

• Polyphenol oxidase catalyzes the oxidation of catechol into orthoquinone, producing dark-colored products.
• The spectrophotometer was used to measure enzyme activity by monitoring the absorbance change over time.
• Reaction rate was determined by calculating the change in absorbance over time.

Factors Affecting Reaction Rate in the Lab

• Substrate concentration: Higher substrate initially increases reaction rate, but levels off due to enzyme saturation.
• Enzyme concentration: Higher enzyme concentration initially increases reaction rate, but eventually levels off due to substrate limitation.

Biological Role of Polyphenol Oxidase

• It plays a role in plant defense by producing quinones, which are toxic to some insects and microbes.

Lecture 6 Lab Review: IV-IX

• IV: Temperature Control: Tested the effects of different temperatures on enzyme activity.
  • Anticipated Results: Optimal activity at 45 degrees Celsius, decreasing at higher and lower temperatures.
• VIII: Cofactors: Investigated the potential role of metal ions as cofactors for polyphenol oxidase.
  • Experimental Design: Incubated the enzyme with chelating agents to remove metal ions.
  • Anticipated Results: Cyanide and PTU (chelating agents) reduce reaction rate, suggesting a copper cofactor.
• IX: Salt Effects: Examined the effect of salt concentration on enzyme activity.
  • Anticipated Results: Increased salt concentration destabilizes enzyme structure and decreases reaction rate.

Gut Microbiome

• The gut microbiome refers to the microbial community inhabiting the large intestine in humans.
• Fiber, undigested by human enzymes, is fermented by gut microbes in the colon.
• Fermentation byproducts (hydrogen, carbon dioxide, and methane in some individuals) are absorbed into the bloodstream and exhaled.

Bristol Stool Score

• The Bristol Stool Scale classifies feces into seven categories based on shape and consistency.
• Stool form is influenced by the gut microbiome's activity.

Freezer Stocks

• Freezer stocks preserve bacterial strains for long-term use.
• Cryoprotectant is added to minimize damage during freezing and thawing.

PCR

• Polymerase Chain Reaction amplifies specific DNA regions, like the 16S rRNA gene.
• Each PCR cycle involves three steps:
  • Denaturation: Separating double-stranded DNA into single strands.
  • Annealing: Primers bind to complementary sequences on the DNA template.
  • Extension: Taq DNA polymerase extends the primer, synthesizing a new DNA strand.

PCR Cocktail

• PCR master mix contains:
  • DNA template: The original DNA to be amplified.
  • Primers: Short DNA sequences that bind to specific target regions.
  • dNTPs: Building blocks of DNA.
  • Taq DNA polymerase: An enzyme that synthesizes new DNA strands.
  • Buffer: Maintains optimal pH for enzyme activity.
  • MgCl2: Cofactor for Taq DNA polymerase.

Enzyme Kinetics

• Vmax is the maximum rate of reaction when all active sites are full and processing substrates.
• Km is the substrate concentration when the reaction rate is half maximal.
• Higher Vmax and lower Km indicate better enzyme efficiency.
• Cofactors are non-protein chemical compounds or metallic ions that help enzymes catalyze biochemical reactions.
• Competitive inhibitors bind to the active site and compete with substrates, maintaining Vmax.
• Noncompetitive inhibitors bind to a different part of the enzyme, changing its conformation and lowering Vmax.
• Percent inhibition helps identify competitive and noncompetitive inhibitors by examining its variation with different substrate concentrations.

Polyphenol Oxidase Lab

• Polyphenol oxidase catalyzes the oxidation of catechol into orthoquinone, producing dark-colored products.
• Spectrophotometer measures absorbance changes over time, reflecting enzyme activity.
• Reaction rate is calculated as the change in absorbance over time, typically using the 0-2 minute data.
• Substrate concentration affects reaction rate by initially increasing it due to reduced finding time, but eventually plateaus as the enzyme becomes saturated.
• Enzyme concentration also increases the initial reaction rate, but eventually levels off as substrate becomes limiting.
• Biological role of polyphenol oxidase includes plant defense by producing quinones, toxic to insects and microbes.

Temperature Effects

• Temperature influences enzyme activity, with optimal rates at around 45 degrees Celsius.
• Low temperatures slow down reactions, while high temperatures can denature enzymes, leading to reduced or no activity.

Freezing/Thawing vs. Boiling

• Freezing and thawing maintain enzyme function, resulting in faster reaction rates compared to control.
• Boiling denatures the enzyme, leading to slower reaction rates.

Potassium Arsenite

• Potassium arsenite reacts with sulfhydryl groups in proteins, disrupting disulfide bonds needed for tertiary structure.
• Arsenite shows minimal or no inhibition of polyphenol oxidase due to the absence of sulfhydryl groups in its structure.

Para-hydroxybenzoic Acid (PHBA)

• PHBA has a similar structure to catechol, suggesting potential inhibitory effects.
• PHBA exhibits partial inhibition of polyphenol oxidase, with greater inhibition at low substrate concentrations, identifying it as a competitive inhibitor.

Metal Ion Cofactors

• Chelating agents remove metal ions from enzyme active sites.
• PTU and cyanide inhibit polyphenol oxidase activity, suggesting copper as a cofactor.
• Cyanide affects both copper and iron, making its role in enzyme activity unclear.

Salt Effects

• High salt concentrations destabilize ionic bonds, leading to denaturation of the enzyme and reduced activity.

Gut Microbiome

• Gut microbiome refers to all microbes present in the gut environment, primarily in the colon.
• Fiber fermentation occurs in the large intestine by gut microbes who break down complex carbs into simpler compounds.
• End products of fermentation include hydrogen, carbon dioxide, and methane, produced by a minority of individuals.
• Breath samples at the end of exhale contain fermentation byproducts diffused from the bloodstream into the lungs.
• Methane production varies between individuals due to differences in microbiome composition.
• Bristol Stool Scale categorizes stool into 7 types based on shape and consistency, reflecting gut microbiome activity.
• Freezer stocks are prepared to preserve bacterial strains for long-term studies.

PCR (Polymerase Chain Reaction)

• PCR amplifies specific DNA regions, like the 16S rRNA gene.
• Three steps of each PCR cycle:
  • Denaturation: Double-stranded DNA separates at 98 degrees Celsius.
  • Annealing: Primers bind to complementary sequences at 50-65 degrees Celsius.
  • Extension: Taq DNA polymerase synthesizes new DNA strands at 72 degrees Celsius.
• PCR cocktail (master mix) contains:
  • DNA template: Target sequence to be amplified.
  • Primers: Short DNA sequences that bind to the target DNA.
  • Taq DNA polymerase: Enzyme that synthesizes new DNA strands.
  • dNTPs: Building blocks for DNA synthesis.
  • Buffer: Provides optimal pH and ionic conditions for the reaction.

Hypothesis vs. Prediction

• A hypothesis is a testable statement that proposes a relationship between variables.
• A prediction is a specific outcome expected if the hypothesis is true.

Positive vs. Negative Control

• A positive control is a group in an experiment that is known to produce the desired effect.
• A negative control is a group that does not receive the treatment or has conditions where no effect is expected.

What is an enzyme?

• Enzymes are biological catalysts that speed up chemical reactions without being consumed in the process.
• Enzymes are proteins that accelerate reactions.
• Not all catalysts are enzymes.

How do enzymes lower the activation energy barrier?

• Enzymes lower the activation energy barrier by:
  • Binding to the reactants (substrates) and stabilizing the transition state.
  • Orienting the substrates correctly.
  • Providing a favorable environment for the catalytic cycle to occur.

Factors that influence catalysis

• Find time (time for an enzyme to bind to the next substrate)
• Processing time (time for the enzyme to convert substrates into products)
  • High substrate concentration decreases find time but processing time remains the same.

Factors that affect reaction rate:

• Enzyme Concentration: Higher enzyme concentration leads to a faster reaction rate, but eventually levels off as the substrate becomes limiting.
• Substrate Concentration: Higher substrate concentration leads to a faster reaction rate until the enzyme becomes saturated.
• Temperature: Optimal temperature maximizes enzyme activity, but extreme temperatures can cause denaturation.
• pH: Optimal pH maximizes enzyme activity, but extreme pH can also cause denaturation.
• Efficiency of the enzyme: Different enzymes have varying catalytic efficiencies.
• Factors affecting enzyme shape: Factors like temperature, pH, and inhibitors can alter enzyme shape and activity.

Vmax and Km

• Vmax is the maximum rate of reaction, achieved when all active sites are saturated.
• Km is the substrate concentration at which the reaction rate is half-maximal.
• A higher Vmax and a lower Km indicate a more efficient enzyme.

Cofactors

• Cofactors are non-protein chemical compounds or metallic ions that assist enzymes in catalysis.
• Cofactors are essential for the proper functioning of many enzymes, helping them reach their full catalytic activity.

Competitive vs. Noncompetitive Inhibitors

• Competitive Inhibitors:
  • Bind to the active site of the enzyme, competing with substrates.
  • Do not change Vmax but increase Km.
• Noncompetitive Inhibitors:
  • Bind to a different site on the enzyme, altering its conformation.
  • Decrease both Vmax and Km.

How to design an experiment to identify competitive vs. noncompetitive inhibitors:

• Add competitive or noncompetitive inhibitors at varying concentrations and measure the Vmax.
• If Vmax is lowered, the inhibitor is noncompetitive.
• If Vmax remains the same, the inhibitor is competitive.

Calculating Percent Inhibition

• Percent inhibition is calculated based on the difference in reaction rate with and without the inhibitor.
• Competitive inhibitors show varying percent inhibition at different substrate concentrations.

Experimental Design Tips

• Prewarm/Pre-cool the enzyme and substrate to ensure temperature stability throughout the experiment.
• Maintain a constant temperature during the experiment.

Freezing/Thawing vs. Boiling a Enzyme

• Freezing and thawing can maintain enzyme activity.
• Boiling denatures the enzyme, leading to a decrease in reaction rate.

Potassium Arsenite

• Potassium arsenite reacts with sulfhydryl groups in proteins, disrupting disulfide bonds and affecting tertiary structure.
• Potassium arsenite is not an inhibitor of polyphenol oxidase as it does not contain sulfhydryl groups.

Para-Hydroxybenzoic Acid (PHBA)

• PHBA is a competitive inhibitor of polyphenol oxidase.
• It has a similar chemical structure to catechol (substrate).
• It shows partial inhibition, with higher inhibition at lower substrate concentrations.

Polyphenoloxidase and Metal Ion Cofactors

• Polyphenoloxidase requires a copper ion cofactor.
• Chelating agents can remove metal ions from the active site, decreasing enzyme activity.

Increasing Salt Concentration

• High salt concentrations can denature enzymes by destabilizing ionic bonds, reducing enzyme activity.

Gut Microbiome

• Gut microbiome refers to all the microorganisms present in the digestive tract, primarily in the large intestine (colon).

Fiber Fermentation

• Humans lack enzymes to digest most dietary fiber.
• Gut microbes ferment fiber anaerobically.
• End products of fermentation include hydrogen, CO2, and methane.

Detection of Fermentation Byproducts in Breath

• Gases produced during fermentation diffuse into the bloodstream and are exhaled.
• Breath samples should be collected at the end of exhalation to ensure they contain fermentation byproducts.

Methane Production

• Some individuals produce more methane due to a microbiome that metabolizes hydrogen to produce methane.

Bristol Stool Score

• Bristol Stool Scale classifies stool into seven categories based on its shape and consistency.
• Stool form is influenced by the gut microbiome and reflects its health.

Freezer Stocks

• Freezer stocks are prepared to preserve bacterial strains for long-term use.
• Cryoprotectants are used to prevent damage during freezing and thawing.

PCR (Polymerase Chain Reaction)

• PCR is a technique used to amplify specific DNA regions (like the 16S rRNA gene) for analysis.
• It involves three steps:
  • Denaturation: DNA is heated to separate double-stranded DNA into single strands.
  • Annealing: Primers bind to complementary sequences on the single-stranded DNA.
  • Extension: Taq DNA polymerase synthesizes new DNA strands, copying the template.
• Each cycle doubles the target DNA region, leading to exponential amplification.

PCR Cocktail (Master Mix)

• Master Mix: Contains the necessary components for PCR:
  • DNA polymerase: Enzyme responsible for DNA synthesis.
  • Primers: Short DNA sequences that initiate DNA synthesis.
  • Nucleotides: Building blocks of DNA.
  • Buffer: Maintains the pH of the reaction.
  • Magnesium ions: Cofactor required by DNA polymerase.