bscL midterm exam pH&buffers and enzymes

How do pH changes affect proteins?

pH preserves protein shape/function. changes in pH alters the hydrogen bonding and forces the protein to change shape. severe pH changes cause denaturation

acid

any substance that increases the free H+ concentration of a solution by donating H+ ions in an aqueous environment. Acidic solutions have a pH value less than 7, where hydrogen ion concentration exceeds hydroxide ion concentration

base

any substance that reduces the free H+ concentration of a solution by either accepting hydrogen ions or donating hydroxide ions

buffer

a substance that minimizes changes in the concentrations of H+ and OH- ions, helping a solution resist changes in pH

what is the role of a buffer in a biological system ?

A buffer is a substance that minimizes changes of H+ and OH- ions, helping a solution resist changes in pH within certain limits

do acids need to have hydrogen on their [chemical] formulas in order to behave as an acid ; explain

A molecule does not need to have hydrogen in its chemical formula to behave as an acid, provided its reaction in solution increases the H+ concentration

how a buffer works

• When an acid is added: The concentration of H+ in the solution increases, and the buffer resists a pH change by combining the additional H+ ions.

• When a base is added: The concentration of H+ in the solution decreases, and the buffer resists a pH change by releasing H+ ions into the solution

pH indicator w/ plants. how is it prepared?

antocyanines, which change color over different pH ranges, depending on source. by chopping up the red beets or red cabbage into small pieces. Then the juice is strained off from the resulting mush, and the mush can also be extracted with water to yield even more of the pigment. The solutions are filtered to remove any remaining plant matter,

red cabbage juice shift from (pH 1-12)…

deep red/pink in acidic to purple in neutral and green/yellow in basic

red cbeet juice shift from (pH 11-12)…

from red to bright yellow

How do pH changes affect enzymes?

every enzyme has an optimal pH where its reaction rate is highest. ex: pepsin requires a strongly acidic environment (pH 1.5–1.6) in the stomach, while trypsin operates in the neutral range

How does pH maintain homeostasis?

To protect against lethal pH changes organisms use buffers.

importance and function of enzymes

enzymes catalyze chemical reactions, sustains metabolism

how do enzymes act in order to speed up the rate of chemical reactions?

1. lowers activation energy

2. An enzyme binds its reactant molecule (substrate) at a specialized region called the active site, forming a temporary enzyme-substrate complex

3. Within the enzyme-substrate complex, the enzyme either:

   1. anabolic: The active site holds substrate molecules in close proximity and proper orientation so they can readily interact

   2. catabolic: changing shape upon substrate binding to strain specific chemical bonds within the substrate the enzyme undergoes shape changes to make those bonds easier to break or modify

4. the enzyme does not form permanent chemical bonds with the substrate and remains completely unchanged by the reaction

the effects of temperature on enzyme activity

As temperature increases, the reaction rate increases (vice versa),

Every enzyme functions best at an optimal temperature,

Denaturation at High Temperatures, lower temp does not denature the enzyme

the effects of pH on enzyme activity

Each enzyme has a specific optimal pH where its catalytic activity is highest,

Deviating from the optimal pH alters the active site to change shape,

Extremely acidic or basic conditions cause denaturation and a complete loss of catalytic function

the effects of enzyme concentration (dilution) on enzyme activity

Adding more enzyme provides more available active sites which decreases the time required to complete hydrolysis or product formation

Diluting the enzyme reduces the number of active sites available, meaning fewer substrate molecules can be processed simultaneously, which slows down the overall reaction rate

If substrate concentration becomes limiting or if enzyme molecules outnumber available substrate molecules, adding further enzyme will no longer increase the reaction rate and the activity curve levels off

competitive inhibitors. is it reversible?

a molecule that is structurally similar to the natural substrate of an enzyme and competes directly with the substrate for a position at the active site which prevents the real substrate from binding and thereby slowing or stopping the reaction rate.

can be reversed by raising the concentration of the substrate to sufficiently high levels while keeping the inhibitor concentration constant

non-competitive inhibitors. can it be reversed?

The inhibitor binds to a region on the enzyme other than the active site which alters the enzyme so that its catalytic properties are lost by either physcially blocking the substrates’ access to the active site or the inhibitor induces a conformational change (change in shape) in the protein, rendering the active site unable to fit or process the substrate

increasing the substrate concentration will NOT reverse non-competitive inhibition

what were the substrates that we used to test for enzymes

Catechol: Used as the substrate for the enzyme catechol oxidase

Starch: Used as the substrate for the enzyme amylase.

What happened when we treated / tested catechol under different denaturing conditions e.g. temp, pH, or dilutions ?

Temperature Treatments:

• 0°C (Ice): Slowed molecular collisions, resulting in very light/slow browning (benzoquinone formation), but the enzyme was not denatured.

• 40°C (Optimal): Produced the darkest brown color in the shortest time, representing the optimal temperature for catechol oxidase activity.

• 100°C (Boiling): Showed no browning (solution remained clear) because extreme heat denatured the enzyme protein, destroying its active site

pH / Acid Treatments:

• Treating potato tissue with citric acid (lemon juice) or vinegar lowered the pH and denatured catechol oxidase by altering amino acid charges and disrupting ionic bonds. Consequently, no benzoquinone was produced and the fruit/vegetable tissue remained unbrowned

How did we test it in the lab for non-competitive/competitive inhibitors?

enzyme: potato extract

substrate: catechol

inhibitor tested: PTU

The positive control tube had a dark brown color, the tube w double the substrate+inhibitor+enzyme stayed clear, and the substrate+inhibitor+enzyme stayed clear as well

What kind of colorimetric reactions should we expect from these assays if the enzyme did work ?  If the enzyme was inactivated ?

catechol:

• active: dark brown

• inactive: clear

amylase

• active: yellow, orange, amber

• inactive: dark blue-black

catabolic reactions

splitting molecules into smaller subunits

anabolic reactions

combining smaller molecules into larger ones

activation energy

the initial investment of energy that must be supplied for reactant molecules to react with one another

What happened when we treated / tested amylase under different denaturing conditions e.g. temp, pH, or dilutions ?

• Temperature Treatments:

  • Optimal Temp (~37°C–40°C): Starch was hydrolyzed into maltose rapidly. samples quickly shifted from dark blue-black to light yellow/orange, signaling complete starch digestion.

  • 100°C (Boiling): Heat denatured amylase. On spot-plate assays, samples remained dark blue-black across all time intervals because the enzyme was inactivated and could not hydrolyze starch.

  • 0°C (Cold): Lowered kinetic energy and reduced molecular collisions, increasing the time required to hydrolyze starch.

• pH Treatments:

  • Optimal pH (~6.7–7.0): Amylase achieved its fastest digestion time in the neutral range.

  • Extreme Acid (e.g., pH 2): Extreme pH caused denaturation. On spot-plate tests at pH 2, the iodine solution remained dark blue-black over time, showing no starch breakdown.

• Enzyme Concentration (Dilution) Treatments:

  • Higher Concentration (Less Diluted): Decreased the time needed for complete hydrolysis because more active sites were available to process substrate molecules simultaneously.

  • Lower Concentration (More Diluted): Increased the time required to clear starch because fewer active sites were present.