Lab Six: Enzyme Kinetics!

We like it fast, give us some enzymes!

The last step of Glycolysis:

PEP + ADP --> Pyruvate + ATP

Enzyme: Pyruvate Kinase

What's important in this lab?

• What is the importance of an enzyme?
• What is Km?
• What is Vmax?
• What does DNPH do?
• What does NaOH do?
• Why do we add the Pyruvate Kinase last?
• What does a Michaelis-Menton Plot show?
• What does a Lineweaver-Burk Plot show?
• Why is time so important?
• What is an activator? an inhibitor? How can you tell?

Enzymes:

The energizer bunny of reactions!

It keeps going and going until there's no substrate left! They lower the activation energy of the reaction so it takes less to get to the final product!

Km-the michaelis constant

This is the measure of the affinity of the enzyme for the substrate.

Km= 1/2Vmax

1/2 Vmax? What's Vmax?

The maximum rate an enzyme can convert a substrate into a product.

There's only so fast the bunny can pound that drum before his arms fall off! That is his Vmax!

What does DNPH do?

Gives the Pyruvate color so as to absorb light during spec at 510nm.

What does NaOH do?

NaOH is a used to alter pH. It alters the solution's pH and stops the

reaction! IMPORTANT! If the reactions are not timed correctly and runs long, more product is made in one reaction than another!!!

We add Pyruvate Kinase last so the reaction starts when the enzyme is added!

Michaelis-Menton Plot

Velocity vs [Substrate]

What is your substrate?

Lineweaver-Burk Plot

1/V vs 1/[S]

This gives a more accurate Vmax (crosses at y-axis) and [Substrate] (crosses at x-axis)

Activators and Inhibitors

What is an activator?

Something that promotes a reaction!

What is an inhibitor?

Something that slows a reaction! Feedback inhibition plays a large role in Enzyme Kinetics!

Let's have story time:

I love to drive! Love it! But this means I have to fill up with gas, ugh! No matter what time of year, I hate standing and holding the gas pump handle!!! So I always put the clip on and walk away. Now why am I telling you about my love for driving and my hate for filling up??

Feedback inhibition is like the automatic click off of the gas pump! The pump is the enzyme, the tank is empty and wants to be filled with our product, gas! The pump continues to produce gas, our product, until the tank reaches capacity and tells the pump to STOP! If the pump did not stop the car would overflow with gas all over the station. The feedback from the tank saying it is full is the same as the product in our Enzyme Kinetics reaction, it tells the enzyme to STOP!