Biochem 4.4 Enzyme Activity Measurement and Analysis

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Questions and Answers

How is enzyme activity typically measured?

Rate of temperature change per unit time

Volume of enzyme solution used per unit time

Amount of substrate consumed per unit time (correct)

Mass of protein in the sample per unit time

What unit is commonly used to express enzyme activity?

U/mg protein

mg/min

μmol/min (correct)

g/min

What is specific activity in regard to enzymes?

Enzyme activity divided by mass of protein (correct)

Total mass of protein in the assay

Rate of substrate consumption per unit time

Total activity divided by enzyme concentration

Why is enzyme concentration important when measuring enzyme activity?

It affects the interpretation of enzyme activity. Signup and view all the answers

How does the measurement of enzyme activity vary?

With the enzyme concentration in the sample Signup and view all the answers

What can lead to an incorrect conclusion in enzyme activity experiments?

Comparing enzyme activities without adjusting for concentration Signup and view all the answers

What is the formula for calculating specific activity?

Enzyme activity / Mass of protein Signup and view all the answers

Which factor does NOT affect enzyme concentration?

Experimental temperature Signup and view all the answers

What is the expected outcome of increasing purification steps on an enzyme's yield?

Yield decreases while purity increases. Signup and view all the answers

Which environmental factor can drastically affect enzyme activity?

Temperature, pH, and salt concentration Signup and view all the answers

In the purification of glycogen synthase from E. coli, what was the activity per mL after affinity chromatography?

7.5 U/mL Signup and view all the answers

How does a higher salt concentration affect the enzyme's structure?

It can disrupt salt bridges. Signup and view all the answers

What is the primary complication associated with inducing expression of enzymes in different cultures?

The enzymes may not fold correctly. Signup and view all the answers

What generally occurs during the purification process of enzymes?

Purity typically increases and yield decreases. Signup and view all the answers

Why is it important to monitor enzyme activity in terms of UDP-glucose consumption?

It assesses the specific activity of the enzyme. Signup and view all the answers

What defines an enzyme's optimal conditions?

The narrow range of pH and salt concentration for peak activity. Signup and view all the answers

Why is it important to divide by total protein mass when calculating specific activity?

To ensure that only the enzyme activity is measured, excluding contaminants. Signup and view all the answers

What is the primary goal of protein purification?

To retain the enzyme of interest while removing contaminants. Signup and view all the answers

What happens to the total activity of the enzyme as purification steps are implemented?

Total activity decreases due to some enzyme loss. Signup and view all the answers

What is the significance of specific activity measurements during purification?

They provide insight into increasing enzyme purity throughout the process. Signup and view all the answers

What does the crude lysate contain that is important for the purification process?

Maximal total activity but numerous contaminating proteins. Signup and view all the answers

How does the presence of contaminants in a crude lysate affect specific activity?

It decreases specific activity since the denominator is large while enzyme activity remains the same. Signup and view all the answers

What is created at the beginning of a protein purification project?

A crude lysate that includes all soluble proteins. Signup and view all the answers

Which step in the protein purification process usually has the highest total activity?

The initial crude lysate sample before any purification. Signup and view all the answers

How is total protein mass calculated?

By multiplying the volume by the measured protein concentration in mg/mL. Signup and view all the answers

What does specific activity measure?

The enzyme activity per unit of protein mass. Signup and view all the answers

What does the fold-increase in purity indicate?

The ratio of specific activity at a certain step to that of the crude lysate. Signup and view all the answers

How is total activity calculated?

Volume (mL) × Activity per mL (U/mL). Signup and view all the answers

Which of the following correctly describes % yield?

The total activity of the purified enzyme divided by the total activity of the crude lysate. Signup and view all the answers

What happens to specific activity with each purification step?

It may increase, indicating higher purity. Signup and view all the answers

What is necessary for the units to cancel out correctly in calculations?

Ensuring that the same units of measurement are applied. Signup and view all the answers

Why is the total activity of the crude lysate important in protein purification?

It serves as a baseline for calculating fold-increase in purity. Signup and view all the answers

What can disruption of salt bridges due to changes in pH or salt concentration lead to?

Enzyme denaturation Signup and view all the answers

How do high temperatures affect enzyme function?

Cause proteins to lose their function Signup and view all the answers

What is the effect of reducing temperature below the optimal range for enzyme activity?

Decrease enzyme-substrate interactions Signup and view all the answers

Why do different enzymes have varying optimal conditions?

Enzymes evolve to suit specific environmental conditions Signup and view all the answers

Which of the following is most likely to disrupt the catalytic mechanism of an enzyme?

Altered pH and salt concentrations Signup and view all the answers

What happens to an enzyme that operates at a pH significantly different from its optimal range?

It may denature or lose activity Signup and view all the answers

What is the significance of physiological conditions for enzymes?

They allow for variation in enzyme conditions within different cellular compartments Signup and view all the answers

How does the surrounding solution's proton concentration impact enzymes?

It influences the ionization of active site residues Signup and view all the answers

What is the optimal pH value for proteins in the cytosol?

7.4 Signup and view all the answers

What is the approximate pH value of the interior of the stomach?

2.0 Signup and view all the answers

Which organelle is likely to have an acidic pH?

Lysosome Signup and view all the answers

How do pathological conditions like acidosis affect enzyme function?

They generally result in suboptimal conditions for enzyme activity. Signup and view all the answers

What effect does fever have on enzyme function?

It can sometimes stress germs but may harm host enzymes. Signup and view all the answers

What are cofactors in relation to enzyme activity?

They are non-protein molecules required by enzymes. Signup and view all the answers

Which type of organisms thrive at moderate temperatures ranging from 20-45 °C?

Mesophiles Signup and view all the answers

What can result from prolonged fever in relation to enzyme activity?

Damage to host enzymes and proteins. Signup and view all the answers

Study Notes

Enzyme Environments and Activity

Enzyme activity varies in different cellular compartments and environments.
Pathological conditions can alter an enzyme's environment, impacting activity.
Enzyme activity is measured by product or substrate consumption per unit time (e.g., µmol/min or enzyme units—U).
Enzyme unit definition can vary based on the system observed.
Enzyme activity depends on enzyme concentration; higher concentration does not always indicate higher activity.
Specific activity is the enzyme activity divided by the protein mass (µmol·min⁻¹•mg protein⁻¹), providing a more relevant comparison.

Enzyme Activity Measurement

Specific activity is calculated as enzyme activity divided by protein mass
The specific activity is reported in units of µmol·min⁻¹•mg protein⁻¹, or simply U/mg protein.
Specific activity is independent of enzyme purity and useful in measuring purity changes during purification.
Measuring Total Protein Mass: Calculated by multiplying the volume by the protein concentration (mg/mL)
Measuring Total Activity: Calculated by multiplying the volume by the activity per ml (U/mL).

Protein Purification

Protein purification aims to remove contaminants while retaining the protein of interest.
Crude lysate contains the highest total enzyme activity, but also many contaminants.
Subsequent purification steps typically reduce total activity, but specific activity increases.
Specific activity increases as the protein of interest becomes a larger percentage of total protein mass.
Purification methods can use different separation techniques to achieve desired purity.

Environmental Conditions and Enzyme Activity

Enzyme activity is affected by environmental conditions such as pH, salt concentration, and temperature.
Enzymes have optimal ranges of these conditions for maximum activity; deviations can cause denaturation.
Conditions like pH and salt concentration affect enzyme structure, stability, and catalytic mechanism.
Temperature affects enzyme activity; optimal temperatures exist, and extreme temperatures cause denaturation.

Cofactors and Coenzymes

Enzyme activity depends on the presence of non-amino acid cofactors like metal ions.
Coenzymes are organic cofactors.
Prosthetic groups—strongly bound cofactors—are essential for enzyme function.
Cofactors and coenzymes play roles in structural support and catalysis.
Some molecules act as cosubstrates (e.g., NAD+, NADP⁺, FAD), changing form during the catalytic process but often regenerated by other enzymes.
Minerals and vitmains are critical cofactors and coenzymes precursors for optimal enzyme function.

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Description

This quiz explores the various methods of measuring enzyme activity, the importance of enzyme concentration, and the factors affecting enzyme functionality. It delves into calculation methods, such as specific activity, and highlights potential complications during enzyme purification. Test your knowledge to understand the intricacies of enzymatic activity!