INSTRUCTOR GUIDE: WRITTEN AND PICTORIAL HOW-TO, LEADING QUESTIONS, NOTES
Creating the Hallmark ‘Steady-State Image’ (Figure 2, main text) How To:
Input the example enzymatic reaction information into the simulation software.
1) Open KinTek Explorer Software on a computer that is projected for all students to view.
2) In the Project box click New. This will automatically open the Model Editor and Edit Model feature that is used to construct the model enzymatic reaction detailed in Scheme 1 of the main text and that should be in plain sight on the whiteboard.
3) Type in the following model (E+S=ES=E+P). Click Create.
4) With student help, input rate constants (k+ =
100 & k- = 10 for the E+S=ES reaction and k+ = 1
& k- = 0 for the ES=E+P reaction) in the Model Editor box by clicking on each pre-existing, default rate constant to open up a text box.
5) Scroll down to the Experiment Editor box and click New to enter the starting reaction concentrations of
enzyme (0.1) and substrate (0.5) (with student help) by
clicking on each pre-existing, default initial concentration to open up a text box.
Leading Questions: What value do I input for the binding rate constant for substrate and enzyme? What value do I input for the dissociation rate constant? What value do I input for the catalysis rate constant? What value do I input for the binding rate constant for product and enzyme? What values should I enter for the initial enzyme and substrate concentrations?
Notes: Make sure students indicate units in their answers even though units are not explicitly entered into the software. The product and enzyme binding rate constant question is the hardest because they have to infer the value of 0 as the step does not contain an arrow. You may want to point out that often kinetic experiments are performed with the goal of calculating rate constants, even though in this exercise these values are already known. Ask multiple times if anyone has any questions or anything is unclear before proceeding. The set-up is vital for them being able to follow and get the most use out of the demonstration.
How To:
Observe time traces of different chemical species.
6) In the Experiment Editor click in the text box to the right of ‘S1’ under the Observables and enter a chemical species to observe (maybe S).
7) Hit ‘tab’ on the computer to display the Observable concentration as a function of time (and see if the student’s predication was accurate). The cursor is automatically positioned in another open ‘S2’
Observables text box.
8) Enter the following chemical species, one at a time, in separate Observablestext boxes: enzyme-
substrate complex (ES), free enzyme (E), product (P), substrate (S), and total enzyme (ES + E).
9) The hallmark ‘steady-state image’ (Figure 2, main text) has now essentially been
created. (That image shows a time scale from 0-2. Time scale discussed in next how to.) Leading Questions: Looking at the reaction we are simulating, what do we expect to happen to the substrate concentration over time? What do we expect the product concentration time trace to look like? What will happen to the total enzyme concentration (E + ES)? What will the individual
time traces of E and ES look like? This program lets us simulate any signal we’d like, however, we may not always be able to observe a signal for each chemical species in an actual
experiment. What is an example of a physical signal we could use to track product formation?
Notes: Typically students will be able to anticipate that the S concentration will decrease and the P concentration will increase over time. Because of this fact, the instructor may want to start by looking at these Observables first. The total enzyme concentration remaining constant during the entire time course is often forecasted properly, though some smaller scaffolding questions are sometimes necessary. The E and ES traces are significantly more difficult to envision and also to describe and questions about them may be omitted or posed and then answered by the instructor. Students also ought to be able to predict where the E and S time traces will start at time zero since the initial concentrations were input into the simulation.
The expected response in my classroom pertaining to the physical signal is that the product may absorb light, since students perform a kinetic experiment during lab where the formation of 4-nitrophenol is monitored via light absorption.
How To:
Change the time scale of the simulation.
10) In the Experiment Editor box, either click on the pre-existing, default initial time to open up a text box and type in a value of 10 or click/hold
on the default initial time and drag upward until a
value near 10 is reached.
Leading Questions: Can you please verbally state what the steady-state assumption is? In your opinion, there are many acceptable answers here, where does
the steady-state begin and end? How about you (a different student), in your opinion, where does the steady-state begin and end? Why might two logical people both arrive at different acceptable answers to this question?
Notes: Make sure you are viewing a time duration that extends past the steady-state when asking students to try and identify the range. Students often need coaxing to finally ‘put themselves out there’ and suggest a beginning and an end to the steady-state phase. Since the steady-state is where the concentration of ES stays approximately the same or unchanging, different people can have varying ideas about what approximately means. One person may be comfortable with only a 5% deviation of ES while another person thinks a 25% deviation is acceptable.
How To:
Lengthen the duration of the steady-state by lowering the initial E concentration.
11) Uncheck the Observables E, P, S, and ES+E so that only ES remains visible.
12) Position the mouse above the initial E concentration of 0.1. Click/hold and drag the mouse down. The numerical value should decrease and the ES time trace should start to ‘flatten’ and ‘shrink’ as the simulation generates data using the decreasing initial E value.
13) Once this important visual signal has been noticed, type in a value of 0.001 for E.
14) Manipulate the observed reaction time to include times past the steady-state (900 will work). See ‘extended footage 1’ in the Supporting Information for a video how-to of this step.
Leading Questions: Now, after lowering the initial enzyme concentration, where does the steady-state begin and end? Would it be easier to gather data during the steady-state portion of reaction with high or with low enzyme concentration? Why?
Notes: For the new steady-state duration determination, stay consistent with what ES staying
‘approximately steady’ meant for one of the student answers in the above section. The steady
state intervals described in the main text result from an ES deviation of about 10% being
‘steady’. It should be easier to gather initial rate data when the steady-state exists for a much longer time.
Creating the Hallmark ‘Initial Rates Image’ (Figure 3, main text) How To:
Observe the initial velocities of reactions with constant initial E and varying S values.
15) Uncheck the ES Observable and recheck the P Observable so that only the product time trace is visible.
16) Add more initial S concentrations (0,0.01,0.05,0.1,0.2,0.5,1,2,5) which give rise to multiple P formation traces of varying colors, corresponding to the different initial S values input. (Ask ‘how many reactions question’ given below.)
17) Change the time to zoom in to where the P formation data are essentially linear for the varying initial S
concentrations (10 would work). The hallmark ‘initial rates image’ (Figure 3, main text) has now been created.
18) Select the aFit option located to the left of the P Observable text box, check the linear fit option, f(t) = b*t + C and click the Perform Fit button. Dark grey lines will appear on the screen and are linear fits to the P formation data. (Ask ‘slope questions’ given below.)
19) Scroll below the Perform Fit button and pick out one of the substrate
concentrations to analyze (maybe 0.5).
Find the value for the slope, indicated as ‘b’ in the above equation (0.00082 if using 0.5 initial S value). Record this value and proper units (µM s-1) on the whiteboard. See
‘extended footage 1’ in the Supporting Information for a video how-to of this step.
Leading Questions: Do all of these different initial substrate values require a new reaction?
Notice how the slope of product formation increases with initial substrate concentration. What do you notice about the slopes, relative to each other, at the highest initial substrate concentrations?
Why do you think this is happening? What are the units of these slopes? Look at the time interval we are observing for product formation, are we in the steady-state range of the reaction?
Notes: Yes, each new initial S value requires a new reaction as it is much easier to simulate than actually generate data. At the highest initial S values, the slopes are essentially the same as the enzyme is becoming saturated. Again, stress that rates have units and use the ‘rise over run’ if needed to remind them. Yes, we are in the steady state range for most of the time trace.
(This assumes you are looking at a proper time interval as noted above.) The earliest time points will technically be before the steady-state begins but get ‘washed out’ by the later time points in the steady-state phase. Each unique reaction (S and E value pair) will have its own unique steady-state duration, with the lower S value reactions having shorter durations.
Creating the Hallmark ‘Hyperbolic Curve Image’ (Figure 4, main text) How To:
Plot the initial reaction rates versus initial S values.
20) Scroll back up and select the Rate vs Conc option that appears right below the Perform Fit button. The linear fits, which represent the initial reaction velocities (µM s-1), are plotted in a pop-up display box as a function of initial S
concentration (µM) to create the hallmark ‘hyperbolic curve image’
(Figure 4, main text).
21) Locate the data point on this graph that corresponds to the slope value written on the board.
Leading Questions: How does this plot of rate versus concentration relate to the last plot we just looked at and is still visible in the background? Rate vs conc., concentration of what exactly?
What’s a decent visual estimate for the vmax value? How could we convert the vmax value into a kcat value? Does our estimated kcat value make sense in light of our example enzymatic reaction?
What’s an approximate visual estimate for the KM value? How does our estimated KM value compare to the KM value we’d get from using the mathematical definition of the KM value?
Notes: If the connection between the initial rates plot and the rate vs concentration plot is still not apparent to the students, match up another rate value with the corresponding data point on the graph. This typically makes the interplay between the two graphs readily apparent.
This should also help them realize that it is the initial concentration of substrate that is the x- axis for the hyperbolic graph. Typically, students respond that a value of 0.001 µM s-1 is the vmax. This leads to a kcat value of 1 s-1 (0.001 µM s-1 / 0.001 µM). This is the rate constant plugged into the simulation for the catalysis rate constant so it makes sense that we arrive at this value. The KM value is more difficult to visually estimate (though student’s young eyes can usually do so without the need to use the zoom function of the pop-up box). An estimated
value of 0.1 µM agrees quite well with the 0.11 µM value that is calculated using the rate constants (1 s-1 + 0.1 s-1 / 10 s-1).
How To:
Highlight that vmax is dependent on the initial E value, but kcat is not.
22) With the rate vs concentration graph and the time traces and linear fits of P formation both visible, click/hold and drag the initial E value to a new value (less than 0.01). The simulated P time traces will change immediately and the Rate vs Concentration graph will update once the mouse click is released. See ‘extended footage 1’ in the Supporting Information for a video how-to.
Leading Questions: What is the relationship between initial E concentration and the vmax value?
Notes: Students should be able to identify that as initial E increases the vmax increases (up to a point since at too high of E values it would be in excess over S) and as initial E decreases the vmax decreases. Some may even notice that the vmax value normalized by the total enzyme concentration (kcat) is unchanging. Hopefully, at the least, any students who were still unsure about the connection between the product formation data and the hyperbolic curve are now comfortable.
