Bio labt test 2: lab 5 enzyme kinetics

What is the reaction catalyzed by catalase?

2 H₂O₂ → 2 H₂O + O₂. It breaks down hydrogen peroxide into water and oxygen gas.

Why is catalase biologically important?

H₂O₂ is a toxic byproduct of cellular metabolism (especially fatty acid oxidation in peroxisomes). Without catalase it would accumulate and damage DNA and proteins.

What class of enzyme is catalase?

Oxidoreductase — it catalyzes oxidation/reduction reactions.

Why do we measure O₂ production instead of H₂O₂ consumption?

O₂ is a gas that displaces water in a burette, giving a direct real-time readout. It's much easier to measure than tracking H₂O₂ disappearance.

What is initial velocity (V₀) and why do we use it?

V₀ is the reaction rate at time zero, measured from the earliest linear part of the O₂ production curve. We use it because substrate concentration is still essentially the starting concentration — later measurements are distorted by substrate depletion and product accumulation.

What shape is the curve when you plot V₀ vs [S], and why

Hyperbolic, because enzymes can be saturated. At low [S] activity rises quickly, but as active sites fill up the rate of increase slows until it plateaus at Vmax.

What is Vmax?

The maximum reaction velocity, achieved when all active sites are occupied (enzyme is saturated). Adding more substrate beyond this point does not increase the rate.

What is KM?

The substrate concentration at which V₀ = ½ Vmax. It is an inverse measure of enzyme-substrate affinity — low KM means high affinity.

What does a low KM tell you about an enzyme?

The enzyme has high affinity for its substrate — it reaches half-maximal activity even at low substrate concentrations.

What is RSD and what threshold defines a precise assay

Relative Standard Deviation = (SD/Mean) × 100%. An RSD below 10% indicates acceptable precision

Why did we run 4 replicates specifically at 66 mM?

To calculate RSD and evaluate the precision of the assay. The 66 mM trial served as the technical replicate set.

Why is precision important in a biochemical assay?

Because you cannot directly measure accuracy in most biochemical assays, high precision paired with low systematic bias is the best approximation of accuracy you can achieve.

What is a negative control and what were ours?

A negative control produces no signal, establishing the baseline. Ours were Control 1 (no substrate) and Control 2/3/4 (no active enzyme — heat, acid, or base treated).

What does the "no substrate" control tell us?

That the enzyme alone produces no O₂ — confirming that all O₂ measured in experimental trials comes specifically from H₂O₂ decomposition.

What does the "no enzyme" control tell us?

That H₂O₂ does not decompose significantly on its own under these conditions — confirming that all O₂ measured is due to catalase activity, not spontaneous decomposition.

Why is boiling the enzyme a better control than just adding water instead?

It's a "no active enzyme" control rather than a "no enzyme" control — it demonstrates that the protein itself is not causing the signal, and also shows that proteins are sensitive to extreme temperature.

Why does heat denature enzymes?

Heat adds vibrational energy that overwhelms the stabilizing forces (hydrophobic interactions, hydrogen bonds, ionic interactions) holding the protein's 3D structure together, causing irreversible unfolding.

Why does extreme pH denature enzymes?

Excess H⁺ or OH⁻ ions disrupt the ionic bonds and hydrogen bonds that maintain the protein's 3D structure, destroying the active site geometry.

What is the difference between optimum temperature and denaturation temperature?

Optimum temperature is where the enzyme works fastest. Denaturation temperature is where the enzyme's structure permanently breaks down. These are two separate concepts — cold slows the enzyme but does not denature it.

Why is catalase stored on ice during the lab?

To slow degradation (bacterial growth, spontaneous unfolding, proteolysis) and keep the enzyme in a consistent condition across all trials. Cold slows all these processes without denaturing the enzyme.

Why add reagents in the order buffer → substrate → enzyme?

Adding enzyme last gives you precise control over when the reaction starts, ensuring accurate timing of your measurements.

What is a competitive inhibitor?

A molecule structurally similar to the substrate that competes for the active site. At high [S], substrate can outcompete the inhibitor, so % inhibition decreases as [S] increases. KM increases but Vmax is unchanged.

What is a noncompetitive inhibitor?

A molecule that binds an allosteric site (not the active site), deforming the enzyme so it cannot function properly. Substrate cannot displace it, so % inhibition stays constant as [S] increases. Vmax decreases but KM is unchanged.

How do you distinguish competitive from noncompetitive inhibition using % inhibition data?

If % inhibition decreases as [S] increases → competitive. If % inhibition stays constant as [S] increases → noncompetitive.

Is sodium azide a competitive or noncompetitive inhibitor of catalase, and how do you know?

Noncompetitive — the % inhibition remained roughly constant (~39–47%) across all substrate concentrations tested, meaning increasing [S] could not overcome the inhibition.

What cofactor does sodium azide target, and which proteins does this affect?

The iron-heme prosthetic group. It inhibits catalase, cytochrome C (ETC), and hemoglobin — all of which require iron-heme to function.

What is the turnover number (kcat)?

The number of substrate molecules each active site converts to product per unit time at maximum capacity. Calculated as Vmax divided by the number of enzyme molecules.

Why is catalase described as a kinetically perfect enzyme?

Its kcat/KM ratio reaches the theoretical upper limit (10⁸–10¹⁰), meaning it converts substrate to product almost as fast as substrate diffuses to the active site — it is essentially limited only by diffusion.

Why were trials at 11 mM and 219 mM excluded from the class saturation curve?

They were identified as outliers likely due to technical error at the extremes of the concentration range, and excluding them keeps the class dataset reliable.

What one source of technical error could cause variation between replicates in this assay?

Loss of solution during pipetting — different volumes of enzyme or substrate between trials change concentrations and therefore reaction rates.

How do you calculate the % activity and of inhibition

For % activity: We do the initial velocity with inhibitor divided by initial velocity without inhibitor times 100%

For % inhibition, we do 100% - % activity