Enzyme Kinetics Lab Protocol

Learning goals for this week’s lab
Students should:

  1. Understand that enzymes act as unchanged catalysts to speed up reactions in cells
  2. Be able to estimate Vmax and Km from a graph of reaction rate vs. substrate concentration.
  3. Understand that rate can be saturated, and that it depends on the concentration of substrate (in the case where [S]>>[E]
  4. Distinguish between competitive and non-competitive inhibitors based on changes in Vmax and Km

Lactose is a disaccharide made of glucose and galactose. It is mostly commonly found in milk (including human milk) so organisms who depend on milk for energy (infants, calves, kittens and other mammalian babies) split it into glucose and galactose. The reaction is greatly aided by an enzyme called lactase.
Lactase in humans is encoded by the LCT gene found on chromosome 2. Infants express lactase, but transcription of the gene declines after weaning, resulting in a condition in adults called lactose intolerance. Some populations continue to express lactase after infancy (called lactase persistence), due to a mutation in the LCT gene promoter. This mutation is believed to have occurred between 5 and 10 thousand years ago, and involves a single base change in the promoter region which prevents binding of the transcription factors that normally down-regulate expression in adulthood. It allows for the enjoyment of ice cream, fresh cheeses and milk.
Conveniently for us, lactose intolerance is common enough in the population that lactase is sold at pharmacies though apparently the marketers have spelling trouble and call it “lactaid.” Silly business majors.
Today we are going to examine the “kinetics” of lactase. That is, we will be measuring the rate of this reaction:

Because it’s very difficult to accurately measure lactose or glucose in solution, we use a false substrate (known as analog) for the enzyme known as ONPG (ortho-nitro-phenyl-galactoside ). ONPG has a structure similar to lactose, so it can bind to the enzyme and be cleaved. The products are galactose and ortho-nitrophenolate; ortho-nitrophenolate is yellow in color and can be measured by spectrophotometry.
Measuring the rate of an enzyme is tricky business. The reaction is usually expressed like this

E is the enzyme, S is the substrate, ES is the complex of the two and P is the product. Each step 1) formation of ES and 2) formation of product goes forward, as well as backwards. The backwards direction increases over time (as the reaction approaches equilibrium). To avoid this issue, we measure enzyme kinetics early in the reaction (well before equilibrium). In our case this will mean after less than 2 minutes. In a normal cell there is a huge excess of substrate over enzyme, therefore the overall rate of our reaction will depend on the amount of substrate and how quickly the enzyme does its job. In terms of our experiment this means that the amount of yellow product in the tube, depends on two things:

  • 1. The amount of lactose in the solution
  • 2. How fast lactase converts ONPG to galactose and ortho-nitrophenolate (enzyme kinetics).

Inhibitors
In the case of lactose intolerance, humans stop transcribing lactase. But enzymes can also be inhibited by chemicals called, surprisingly, “inhibitors.” For instance ibuprofen inhibits cyclooxygenase, thus reducing pain, inflammation and fever. Inhibitors may be endogenous - part of the natural regulation of enzyme activity in the cell or they may be exogenous - drugs that are designed to regulate abnormal enzyme activity.
There are two categories of inhibitors: competitive and noncompetitive.
In competitive inhibition, the inhibitor resembles the chemical structure of the true substrate, and it competes with the substrate for binding to the active site. The inhibitor is only able to bind the free enzyme, and cannot bind the substrate-enzyme complex. Since whether the chemical reaction occurs or not is based solely on whether substrate binds or inhibitor binds (they compete, Km increases), adding substrate in large amounts until there is more substrate than inhibitor will eventually return the rate of reaction to normal (Vmax is unchanged).
In non-competitive inhibition, the inhibitor binds to the enzyme at a site different than the active site. Binding to this site, however, changes the overall shape of the enzyme, slowing the processing of substrate into product once it is bound to the enzyme. This form of inhibition does not prevent the binding of the substrate (Km is unchanged) but the reaction rate at high levels of substrate is decreased (Vmax is lower).

Protocol

Once you have reviewed this protocol, you may find it helpful to follow the brief protocol linked at the bottom of the page.

Making an enzyme stock solution and measuring its activity
First we need a way to relate Absorbance reading from the spectrophotometer (depth of yellow color) to amount of ortho-nitrophenolate product in the tube. Different molecules absorb different wavelengths, ortho-nitrophenolate absorbs at 420nm wavelength. In order to make that correlation, you would need to measure a series of known dilutions of ortho-nitrophenolate, measure them with the spectrophotometer. We have done this for you.
We measured the Absorbance for known amounts of ortho-nitrophenolate, and plotted Absorbance versus concentration. This is known as a standard curve. Using a standard curve you can convert your Absorbance readings into concentrations of product. This will be useful in determining the rate of reaction for the Lactase enzyme in different conditions (amount of product/time = rate).

Part 1: Making the Lactase stock solution

  • Take one pill of Lactaid, and crush it into powder with a mortar and pestle.
  • Add the powder to 5 mL of 0.1 M Phosphate buffer (pH7) and dissolve.
  • Then centrifuge at 3000 rpm for 5 minutes to pellet out the insoluble material.
  • The liquid portion is your enzyme stock.

At this point the amount of lactase activity in your stock is unknown, so it would be good to determine a proper concentration of lactase to use for the rest of your experiments. Usually a 1:1,000 dilution is a good concentration of enzyme to carry out the experiments. To do this you'll make a series of dilutions of this first stock and test them. Make a set of 4 serial 1:10 dilutions and label them as 1:10; 1:100; 1:1,000 and 1:10,000. This will give you a set of stock enzyme solutions of different concentrations. It’s important to keep all solutions for the duration of the experiments in case something goes wrong and you need to back up. So always label your tubes clearly and maintain a "backup" rack of anything you think you are "done" with, just in case you're not really done.

Table 1: Example of a ten-fold serial dilution

When measuring the kinetics of an enzyme, we try to work with relatively low concentrations. However, it’s important to choose a concentration that is neither too dilute (hard to measure activity if there isn't much enzyme) and not too concentrated (spectrophotometer has ceiling limit, after which it can't differentiate say very very yellow from very very very yellow).

To test the activity of the each dilution, make a separate reaction tube for each stock and in that reaction tube add:

  • 3.5 mL of buffer
  • 0.5 mL of ONPG (substrate)
  • 0.5 mL of dilution stock 1:1000 (enzyme)
    Tips:

  • Generally its best to add enzyme last, as this starts the reaction
  • it’s important to note the time the reaction starts and when you have a nice yellow, you will use this time for all of your reactions.
  • Also it can be helpful to stagger the reactions so they don't all finish at the same time, and you're unable to stop them all at the right time.

    Watch the tube carefully, and when a pale yellow color appears in the tube, mark the time it’s been reacting and add 0.5 mL of sodium carbonate to stop the reaction. Measure Absorbance of the yellow color in the reaction tube to determine amount of product and then using the reaction time calculate a rate of reaction (amount of P/time in seconds). Use the dilution that gives you a reasonable amount of time; a minute or two is good, thirty seconds is too short and 5 minutes is too long..

    TroubleShooting
    If it becomes yellow too fast , it means the concentration of your 1:1000 dilution is too concentrated. If this is the case, use your 1:10000 dilution for your experiments.


    If your 1:1000 dilution is too pale or takes a really long time to show color, then it’s not concentrated enough. For this situation, use your 1:100 dilution for your experiments.

    Part 2: Calculate Vmax and Km for Lactase and ONPG
    Now that you have a good working concentration of Lactase, you need to calculate the two main measurements of enzyme kinetics: Vmax (maximum rate of reaction for this enzyme and this substrate under these conditions) and Km (the Michaelis constant, the concentration of substrate that gives one half Vmax – it’s an indirect measure of the affinity of the enzyme for the substrate).

    • In order to determine Vmax, and Km, you should graph Rate (o-nitrophenolate/time) versus Substrate (ONPG) concentration.
    • Vmax, is the maximum rate possible (high substrate concentration)
    • Km is the substrate concentration at 1/2Vmax.

    In order to graph Rate (amount of product made (ONP) over time) versus Substrate Concentration (amount of ONPG), you need to react different amount of ONPG with your enzyme solution that you made, different concentrations of ONPG will be provided.

    • Set up a series of tubes in which the enzyme amount is the same, but the amount of ONPG varies.
        Reaction Set Up:
      • 3.5mL of buffer
      • 0.5mL of ONPG solution (each reaction will use a different ONPG concentration)
      • 0.5mL of enzyme stock solution
    • Run each reaction for the same amount of time.
    • Stop the reactions with sodium carbonate and then measure the Absorbance on the spectrophotometer.
    • You can then use the standard curve (above, and note, it’s a line) to get the approximate concentration of ONP product.
    • To get rate, divide the nmoles of product made by the amount of time you ran the reaction.
    • Graph this versus the ONPG concentration in each reaction.
    • Vmax is the highest rate that is the same for at least two different concentrations of ONPG.
    • Km is the substrate concentration at 1/2 Vmax.

    Part 3: Testing Inhibitors
    Now that you know the basic kinetic parameters (Vmax and Km) of Lactase/ONPG reaction at room temperature, buffer pH 7 and no inhibitors, you can design an experiment to test the effect of inhibitors on Vmax and Km.

    Please be sure to design the experiment and sketch it out on paper before beginning. Clear your plan with your TA. You must have negative and positive controls.

    We have a range of monosaccharides and disaccharides (glucose, lactose, galactose and sucrose) that can be tested to see if they inhibit the ability of Lactase to convert ONPG to o-nitrophenol (ONP). Your groups should choose one inhibitors to test. Your goal is to figure out whether the inhibitor is competitive or non-competitive (see above for how you can tell the difference).

      You will have the following materials available:
    • Enzyme: Lactase
    • Substrate: ONPG, various concentrations provided
    • Potential inhibitors: Sucrose, Lactose, Galactose, Glucose

    NOTE: in order to add the inhibitor to the reaction you should decrease the amount of media in the reaction to make up for what you are adding for inhibitors. This is important to make sure the concentration of enzyme is the same in both the no inhibitor and the plus inhibitor reactions. About 0.5 ml of a potential inhibitor per tube should be enough to show an effect. If not you can try 1 ml.

    Assignment due 11:59 PM day of your lab during the week of 10/21 (10 pts)

    Turn in an Enzyme Kinetics graph for the no inhibitor and inhibitor experiment, the Km and Vmax should be labeled as should the X and Y axes (3 pts each): The graphs should have titles. Next name the inhibitor you chose and whether it was a competitive or non-competitive inhibitor. You should also describe why you came to this conclusion (4 points)

    Safety Notes:

    • As we are working with chemicals, you must have gloves/eye protection and a lab coat (ON, not just near you).
    • Pipettes and glass trash GO IN THE GLASS TRASH, not the regular trash.
    • Chemical reactions go in the chemical waste bottles near the sink after lab.
    • Please do not touch keyboards/doors with gloves on.... and definitely NEVER leave the lab and wander about the hallways/restrooms with gloves on. The gloves protect you, but if you touch things with them on, you get chemicals on surfaces that people without gloves may contact.
    • The centrifuge consists of a rotor, with wells for holding tubes containing the liquid to be centrifuged, a motor to spin the head, speed and time controls, and a brake. To avoid vibration, the centrifuge must be balanced when loaded. This is done by placing tubes containing equal volumes of liquid in opposite wells of the rotor. If the centrifuge begins to vibrate excessively, and sounds funny, stop it and check to make sure the tubes are balanced properly.
    • Note there are two types of test tubes: big and small. The big tubes are at your bench; the small tubes are next to the spectrophotometer. The big tubes should be used for the making stock solutions, for running reactions and centrifuging (they are too big for the spectrophotometer); the small ones are only for spectrophotometer readings (they don't fit in the centrifuge...too small).
    • Be sure not to cross contaminate solutions with your pipettes. If you label your pipettes, you are less likely to accidentally mix one solution into another. If you are measuring out different dilutions of the same chemical, you may use one pipette, but always measure/transfer the most dilute solutions first, stronger concentrations last.
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