E 10

What is an enzyme?

A biological catalyst, usually a protein, that speeds up a chemical reaction without being destroyed in the reaction.

What is a substrate?

The compound that an enzyme acts upon.

What is a product?

The substance formed after an enzyme acts on its substrate.

What happens when an enzyme binds its substrate?

An enzyme-substrate complex forms, allowing the substrate to be converted into product.

Is an enzyme destroyed during a reaction?

No. The enzyme is released after product formation and can bind another substrate.

What is the general form of an enzyme-catalyzed reaction?

Substrate --enzyme--> Product.

What enzyme was studied in Experiment 10?

Acid phosphatase from wheat germ.

What was the substrate in the acid phosphatase assay?

Nitrophenyl phosphate.

What products were formed when acid phosphatase acted on nitrophenyl phosphate?

Nitrophenol and free phosphate.

What reaction did acid phosphatase catalyze in this experiment?

Acid phosphatase broke down nitrophenyl phosphate into nitrophenol and free phosphate.

What does acid phosphatase normally do?

It removes phosphate groups from molecules.

Where did the acid phosphatase used in this experiment come from?

Wheat germ extract.

What was used to extract acid phosphatase from wheat germ?

An extraction buffer containing NP-40.

What is enzyme reaction velocity or reaction rate?

The amount of substrate consumed or product formed per unit time.

How was enzyme activity measured in Experiment 10?

By measuring the amount of nitrophenol product formed over time.

What is V0?

The initial velocity or initial reaction rate of an enzyme-catalyzed reaction.

How is V0 determined from a product-versus-time graph?

V0 is the slope of the early linear portion of the graph.

What are the units of V0 in this experiment?

nmoles of nitrophenol produced per minute (nmole/min).

Why do we use the early part of the reaction to calculate V0?

Product formation is approximately linear with time during the early part of the reaction.

Why does an enzyme reaction rate decrease as time passes?

Substrate and/or required cofactors become depleted, causing product formation to slow down.

What happens to a product-versus-time graph as the reaction begins to slow?

The graph begins to curve or flatten instead of remaining linear.

Which data points should be used for linear regression when calculating V0?

Use as many points as possible from the early linear portion of the product-versus-time graph.

Should later points that cause the product-versus-time graph to curve be used to calculate V0?

No. V0 should be determined from the linear portion of the reaction.

Memory trick for V0?

V0 = Velocity at the Very beginning.

How does increasing the amount of enzyme affect V0?

V0 increases as the amount of active enzyme increases.

What is the relationship between enzyme amount and V0?

V0 is proportional to the amount of active enzyme.

If you double the active enzyme concentration while substrate is available, what should happen to the initial reaction rate?

The initial reaction rate should increase because more enzyme molecules are available to catalyze the reaction.

How does temperature affect enzyme activity?

Increasing temperature initially increases reaction rate, but excessive temperature denatures the enzyme and decreases activity.

What happens to enzyme reaction rate as temperature increases up to about 45°C?

The reaction rate generally increases.

What happens to many enzymes at very high temperatures?

They denature and become inactive.

At approximately what temperatures does the procedure say enzymes are usually denatured and inactivated?

About 50–70°C.

What is a temperature optimum?

The temperature at which the enzyme has its maximum reaction rate.

How does pH affect enzyme activity?

Each enzyme has an optimum pH; moving too far above or below that pH can decrease activity or denature the enzyme.

What was the optimum pH of acid phosphatase in this experiment?

pH 4.5.

Why is it called acid phosphatase?

It has a low, acidic pH optimum.

What happens to acid phosphatase at alkaline pH?

It becomes catalytically inactive.

How does increasing substrate concentration generally affect enzyme reaction rate?

Increasing substrate concentration generally increases reaction rate until the available enzyme molecules become saturated.

Why does increasing substrate eventually stop increasing enzyme reaction rate?

At high substrate concentration, the available enzyme active sites become occupied, so enzyme amount becomes limiting.

What factors can influence enzyme activity?

Amount of enzyme, amount of substrate, temperature, and pH.

What was the purpose of the nitrophenol standard curve?

To convert OD410 absorbance readings from the enzyme assay into the concentration of nitrophenol produced.

What were the two variables on the nitrophenol standard curve?

Nitrophenol concentration and OD410 absorbance.

What goes on the x-axis of the nitrophenol standard curve?

Nitrophenol concentration in nmole/ml.

What goes on the y-axis of the nitrophenol standard curve?

OD410 absorbance.

What wavelength was used to measure nitrophenol?

410 nm.

What does OD mean?

Optical density.

What concentrations of nitrophenol were used for the standard curve?

0, 12.5, 25, 50, 100, and 200 nmole/ml.

How were the nitrophenol standards prepared?

By serial dilution from the 200 nmole/ml nitrophenol standard.

Which standard tube was used as the blank?

S1.

What was the concentration of nitrophenol in S1?

0 nmole/ml.

What was the concentration of nitrophenol in S6?

200 nmole/ml.

What was added to all six standard-curve tubes before measuring OD410?

1 ml of KOH.

What should a standard curve graph include?

A title, properly labeled axes with units, a linear regression equation, and R² value.

What should be done with the y-intercept of the standard curve in this experiment?

Set the y-intercept to zero.

What should you do if the standard curve has an R² less than 0.95?

Identify and drop an outlying data point if appropriate and recalculate the regression.

What does a high R² indicate?

The data fit the linear regression line well.

How is an unknown nitrophenol concentration calculated from the standard curve?

Substitute the measured OD410 into the standard curve equation and solve for nitrophenol concentration.

If the standard curve equation is y = mx, what does y represent?

OD410 absorbance.

If the standard curve equation is y = mx, what does x represent?

Nitrophenol concentration in nmole/ml.

If y = mx, how do you solve for nitrophenol concentration?

x = y/m.

How did the lab convert nitrophenol concentration in nmole/ml into amount of nitrophenol in nmoles?

Multiply the concentration in nmole/ml by the volume measured in ml.

What volume was measured at each time point?

1 ml.

If the standard curve gives 50 nmole/ml nitrophenol and the measured volume is 1 ml, how much nitrophenol was produced?

50 nmole.

What is the second major graph used in this experiment?

A graph of nitrophenol produced versus reaction time.

What goes on the x-axis of the product-versus-time graph?

Reaction time in minutes.

What goes on the y-axis of the product-versus-time graph?

Nitrophenol produced in nmoles.

What is the purpose of the product-versus-time graph?

To determine the enzyme reaction rate or V0.

How is the product-versus-time graph produced experimentally?

Run the enzyme reaction, stop samples at different times, measure OD410, use the standard curve to convert OD410 into nitrophenol produced, and graph nitrophenol versus time.

How is V0 obtained from the product-versus-time graph?

Perform linear regression on the early linear data points; the slope of the line is V0.

If the equation of the product-versus-time graph is y = 7.2x, what is V0?

7.2 nmole/min.

What is the main difference between the two graphs in Experiment 10?

The standard curve converts absorbance into product concentration, while the product-versus-time graph determines the enzyme reaction rate.

Standard curve: what is X and what is Y?

X = nitrophenol concentration (nmole/ml); Y = OD410 absorbance.

Product-versus-time graph: what is X and what is Y?

X = time (min); Y = nitrophenol produced (nmole).

Memory trick for the two graphs?

Standard curve = Concentration → Absorbance; Enzyme graph = Time → Product.

What temperature was used for the acid phosphatase enzyme assay?

37°C.

What pH was the nitrophenyl phosphate substrate solution?

pH 4.5.

How much substrate solution was placed into reaction tube A*?

7 ml.

How much acid phosphatase extract was added to reaction tube A*?

50 µl.

Approximately how much pure enzyme did the 50 µl acid phosphatase sample represent?

About 1 µg of pure enzyme.

What started the enzyme reaction?

Adding acid phosphatase to the nitrophenyl phosphate substrate.

What were the reaction time points in the acid phosphatase assay?

0, 5, 10, 15, 20, and 25 minutes.

What tubes were used for the enzyme assay time points?

A1 through A6.

Which tube represented the zero-time sample?

A1.

What was placed into tubes A1-A6 before reaction samples were added?

1 ml of KOH.

What are the TWO functions of KOH in the acid phosphatase assay?

1) Stop the enzyme reaction by making the solution alkaline, where acid phosphatase is inactive; 2) cause nitrophenol to turn yellow.

Why does KOH stop the acid phosphatase reaction?

Acid phosphatase is catalytically inactive at alkaline pH.

Why does KOH help us measure the product?

KOH makes the nitrophenol product yellow so its amount can be measured at 410 nm.

What does increasing yellow color indicate?

More nitrophenol product has been produced.

Why should the pipette tip not touch the KOH in A1-A6 when removing samples from A*?

KOH could contaminate the reaction mixture, raise its pH, and stop/inhibit the acid phosphatase reaction prematurely.

After adding enzyme, what was done with the A* reaction tube?

It was placed in a 37°C water bath and the timer was started immediately.

What happened at 5, 10, 15, 20, and 25 minutes?

1 ml of reaction mixture was removed from A* and placed into the corresponding KOH-containing tube.

Why was A1 collected immediately after mixing enzyme and substrate?

It represented the zero-time value of the reaction.

Which sample was used to autozero the spectrophotometer for the enzyme assay?

A1.

Which samples were measured at OD410 after autozeroing?

A2 through A6.

Describe the complete acid phosphatase reaction.

Acid phosphatase + nitrophenyl phosphate → nitrophenol + free phosphate.

Describe the acid phosphatase assay in one sequence.

Mix acid phosphatase with nitrophenyl phosphate at pH 4.5 → incubate at 37°C → remove samples at different times into KOH → KOH stops the reaction and turns nitrophenol yellow → measure OD410 → use the standard curve to determine nitrophenol produced → graph product versus time → slope of the linear portion = V0.

What are common errors when drawing the standard curve?

Switching the axes, missing units or labels, including the wrong variables, failing to set the intercept to zero, or failing to include the equation and R² value.

What are common errors when drawing the product-versus-time graph?

Switching the axes, graphing OD410 instead of converted nitrophenol amount when asked for product, missing units, and using nonlinear late reaction points to calculate V0.

How can you tell whether a student calculated V0 incorrectly from a graph?

If they used the entire curved or plateau portion instead of the early linear portion, or reported something other than the slope as V0.

Why can't OD410 readings alone directly give V0 in nmole/min?

OD410 must first be converted to nitrophenol concentration using the standard curve, then converted to nitrophenol amount and plotted versus time.

What is the overall purpose of Experiment 10?

To measure the activity and initial reaction rate of wheat germ acid phosphatase by following the production of nitrophenol over time.