Scribd
Upload
25 views

Exp't. 206: An NMR Study of Enzyme Activity

This document describes an experiment using NMR spectroscopy to monitor the hydrolysis of N-acetyl-L-methionine catalyzed by the enzyme acylase I. Five solutions were prepared with varying concentrations of acylase I and incubated for 45 minutes to allow the reaction to proceed before stopping it with heat. NMR spectra were then collected on the samples to monitor changes in the methyl group signals of the substrate and products over time and with increasing enzyme concentration in order to determine the percentage of hydrolysis.

Uploaded by

lovehope
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
25 views

Exp't. 206: An NMR Study of Enzyme Activity

This document describes an experiment using NMR spectroscopy to monitor the hydrolysis of N-acetyl-L-methionine catalyzed by the enzyme acylase I. Five solutions were prepared with varying concentrations of acylase I and incubated for 45 minutes to allow the reaction to proceed before stopping it with heat. NMR spectra were then collected on the samples to monitor changes in the methyl group signals of the substrate and products over time and with increasing enzyme concentration in order to determine the percentage of hydrolysis.

Uploaded by

lovehope
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 2

3/ 16/ 92

Exp't. 206
An NMR Study of Enzyme Activity
Adapted by R. E. Krull, M. Cocco, A. Freyer and R. Minard from "An NMR Study of Enzyme Activity" by K. E. Peterman, et al., J. Chem. Ed., 66 (1989), 875-6. Revised
2/20/94.
Introduction
N-acyl derivatives of methionine are the most susceptible of all substrates toward hydrolysis in
enzymic reactions catalyzed by acylase I. One such substrate, N-acetyl-L-methionine serves as a
good model for demonstrating the use of NMR in enzyme studies. The N-acetyl-L-methionine is
hydrolyzed to produce acetic acid and L-methionine according the the net equation:
CH
3 N
OH
S
CH
3
CH
3
N
OH
S
CH
3
H
O
O
H
O
O
OH
H
H
H
1
2
2
1
N-Acetyl-
L-Methionine
Methionine
Acetic
Acid
+ H
2
O
Acylase I
The chemical shifts of S-methyl 1 (methionine) and acetyl methyl 2 change upon hydrolysis. By
integrating these NMR signals, you will be able to monitor the percent hydrolysis of N-acetly-L-
methionine catalyzed by various concentrations of enzyme. The NMR analysis will be done on the
200 MHz system in Chandlee Lab with an instructor's help. In this experiment, D
2
O (expensive!) is
substituted for H
2
O to minimize the large OH peak in the NMR and to provide the deuterium lock
signal.
Prelaboratory Excercise:
Which NMR signal, the S-methyl 1 (methionine) or the acetyl methyl 2, would you expect to change
the most in the course of this reaction? Explain why.
Using an NMR correlation table, predict the chemical shift for these two signals.
CAUTIONS:
Potassium hydroxide (KOH) is very corrosive. Avoid contact with skin. D
2
O is very expensive.
Only use the amounts called for in the procedures.
Procedure
Two members of the team should collectively prepare the buffer, and then prepare and adjust the
pH the enzyme solution (Acylase 1). The other team members should prepare KOH/D
2
O for
neutralization, and then prepare and neutralize the N-acetyl-L-methionine solution.
With warm soapy water, clean the following glassware: a 50-mL Erlenmeyer flask, 100-mL graduated
cylinder, a 25-mL beaker, a 100-mL beaker, a 25-mL volumetric pipet (from the stock room), and a
glass stir rod. Rinse with acetone and let dry in an oven for 20 minutes. Rinse two 1-mL syringes with
acetone several times, and let them air dry. When using the pH meter (Rm 206), make sure that it is
properly calibrated by checking it with both pH 4 and pH 7 buffers.
Get 4 or 5 NMR tubes from the stockroom.
Prepare a boiling water bath to accomodate the NMR tubes.
In the 100 ml beaker, make 40 ml potassium phosphate buffer (pH=7) as directed below.
Preparation of buffer solution. Weigh 544 mg of potassium phosphate, monobasic (also called
potassium dihydrogen phosphate; formula KH
2
PO
4
; found on the Advanced Shelf) in a 100 mL
beaker and add 40 mL of D
2
O. Stir until it all dissolves. Adjust the pH to 7.0 by dropwise addition of
conc. KOD made by dissolving several KOH pellets in 1/2 mL D
2
O. The pH meter is in lab 216.
2
Preparation of N-acetyl-L-methionine solution. Weigh 120 mg N-acetyl-L-methionine into a 25- mL
beaker, add 10 mL buffer and swirl to dissolve. Using the pH meter, adjust the pH of this solution to
pH 7 using the KOH/D
2
O base made previously.
Preparation of Acylase enzyme solutions. Weigh 10 mg acylase I (720 units/mg) into a 50 mL
Erlenmeyer flask. Rinse a 25-mL pipet with 2 mL of buffer, and then transfer 25 mL buffer to the flask
containing the enzyme, gently swirl to dissolve. To make dilutions required, each team member
should measure out the appropriate volume (see table below) of enzyme stock solution with a
syringe and put it into an NMR tube which has been labelled A to E. Then dilute as required with
buffer. Each team member should take one of these concentrations. One team member may have to
do two.
Dilutions: A. 0.20 mL Acylase sol'n. plus 0.80 mL buffer
B. 0.40 mL Acylase sol'n. plus 0.60 mL buffer
C. 0.60 mL Acylase sol'n. plus 0.40 mL buffer
D. 0.80 mL Acylase sol'n. plus 0.20 mL buffer
E. 1.00 mL Acylase sol'n. plus 0.00 mL buffer
Hydrolysis Reaction. Transfer 1.0 mL of the N-acetly-L-methionine stock solution by syringe to each
of the five NMR tubes. Record the time and let incubate for 45 minutes. After 45 minutes, stop the
reaction by heat denaturing the enzyme: i.e., by placing the NMR tube in boiling water for 10 min. If
the solutions appear cloudy or precipitate is noticed, centrifuge those samples and return the
supernatant to the NMR tube.
NMR Samples. These NMR samples will be run on the 200 MHz system in Rm 7 Chandlee or the
NR-80's in Rm 206 Whitmore. Your instructor will oversee running the samples.
Final Report:
On the basis of the NMR spectra and the methyl singlets that are changing, tell which is due to the N-
acetyl methyl, which is due to the acetic acid that is produced, and which are due to the S-methyl
groups. From the integration of the N-acetyl methyl and acetic acid singlets (one of which is decreasing
and the other of which is increasing), plot a graph with the different concentrations of acylase as the X-
axis and the percentage of hydrolysis as the Y-axis. Remember:
% hydrolysis =
(integral of acetic acid methyl peak) x 100%
(integral of acetic acid + N-acetyl methyl)

You might also like