Optimizing the kinetics experiment

The reaction catalyzed by cytathionine b-lyase is:

CBL will also catalyze a similar b-lyase reaction with cystine and djenkolic acid:

 NH4+

The free thiol produced in each reaction (R-SH), will spontaneously and instantaneously react with 5, 5’-dithiobis(2-nitrobenzoic acid) (DTNB) present in excess in the reaction mixture.

The 5-thio-2-nitrobenzoic acid (5-TNB) product of this second reaction is yellow in color and absorbs visible light at 412 nm.  This quantitative reaction allows us to determine the rate of enzymatic conversion of cytathionine, cystine and djenkolic acid into their respective products.

This week we want to:

1) Calibration curve

Construct a calibration curve, so that we can correlate the concentration of free thiol produced [RSH] and A412.  We’ll use cysteine (stock solution 100 mM) as our reference thiol.

Use the stock solution to make up 6 cuvettes with cysteine concentrations in the range of 0 –100 mcM. Each cuvette should contain 1 mL with Kinetics buffer (200 mM Tris-Cl, pH 9.0), 50 mcL of 5 mM DTNB and a known concentration of cysteine.  Mix well and measure the A412 of each cuvette using the zero cysteine cuvette as the blank.  The extinction coefficient is the slope of a linear regression on a plot of A412 vs. [cysteine].  Units should be A/M or A/mM.

2) Enzyme concentration

Determine an appropriate dilution of the stock enzyme (the tube of enzyme you’ve isolated) to use for subsequent assays.

Make 10 fold, 50  fold and 100 fold dilutions of your wild type and mutant enzymes in protein dilution buffer.  For each enzyme concentration, make up a cuvette with 1.0 mL of Kinetics buffer (pH = 9), 100 mcL of 2.5 mM cystine, 50 mcL of 5 mM DTNB. For each enzyme concentration zero the instrument, then start the reaction by adding 10 mcL of enzyme to the cuvette.  Mix well.  Record the A412 after 1 minute, 2 minutes, 3 minutes 4 minutes and 5 minutes.  Repeat with mutant.  As a control, prepare a cuvette with no enzyme, record the A412 after 1 minute, 2 minutes, 3 minutes 4 minutes and 5 minutes.

Remember we are looking to use the change in absorbance as a function of time to understand the reaction catalyzed by the enzyme.  If the change you observed was only 1-2% ask yourself, is this a good enzyme concentration?  What is the change if no enzyme is added?

3) Calculations (notebook)

Calculate the extinction coefficient of 5-TNB at 412 nm? (A412/M)

Prepare plots of [P] vs time

Using excel, plot the absorbance data collected above as a function of time.  Compare the effect enzyme concentration has on the rate of reaction.  Observe the shape of the curve (or line).  Determine from these plots the "correct" concentration of wildtype and mutant CBL to use in subsequent assays for activity.

Calculate the specific activity of the wild type and mutant CBL you have isolated

A reasonable way to define how active an enzyme preparation is is to determine the “specific activity”, the number of units of activity per mg of protein. If we define a “unit of activity” as the amount of enzyme required to form 0.1 mmol of free thiol in one minute under the reaction conditions used (1.0 mL of Kinetics buffer (pH = 9), 100 mcL of 2.5 mM cystine, 50 mcL of 5 mM DTNB), then what is the specific activity – units/mg –  of your purified CBL?

For comparison, you should also calculate both the specific activity of wildtype and mutant CBL.

How much has the mutation affected CBL activity?

The best way to compare two different enzymes is to consider both the rate enhancement (kcat) and the binding affinity of the substrate for the particular enzyme (Km).  This comparison of catalytic efficiencies can be accomplished by calculating kcat/Km for the wild type and the mutant.  Calculation of kcat and Km requires data to be generated at multiple substrate concentrations.

Today we can compare specific activities.  What is the ratio of wild type activity to mutant activity?

4) Steady state kinetics (as time permits)

Collect initial velocity (Vo) data at pH = 9 using cystine at a variety of [S].

Based on your results from part (2), choose an enzyme concentration that gives reasonable absorbances and record the change in A412 as a function of time using a wide variety of substrate concentration. Use the kinetics method for the Cary 50 spectrophotometer as outlined in the “methods” manual.

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