Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Appendix 3: A Simple
Demonstration of Enzyme
Catalysis
An important (and enjoyable) part of chemistry is to be able to demonstrate
chemical principles by experiment. Many of the experiments described in this
book are highly technical, but in principle enzymes can be easily isolated from a
variety of natural sources. For readers who would like to observe enzyme
catalysis at Wrst hand, I include a brief procedure for a second year experiment
that I set up at Southampton University in 1996, which involves an esterase
activity easily isolated from orange peel!
The experiment involves the regioselective enzymatic hydrolysis of hydroxybenzoic
acid diester derivatives to either the hydroxy-ester or the acyl-acid
product (see Figure A3.1). The para-substituted diester methyl 4-acetoxybenzoate
is commercially available (Aldrich). Alternatively, diester derivatives of
para-, meta- orortho-hydroxybenzoic acid can be readily synthesised by acidcatalysed
esteriWcation of the carboxyl group, followed by pyridine-catalysed
acylation of the phenolic hydroxyl group (see Figure A3.2; see Vogel for
standard procedures). The enzymatic hydrolysis of each diester derivative is
then assayed against orange peel esterase (as a crude extract) and commercially
available (Sigma) pig liver esterase (PLE) and porcine pancreatic lipase (PPL),
using thin-layer chromatography to monitor the appearance of one or other
hydrolysis product and to measure the rate of hydrolysis.
CO 2 Me
CO 2 Me
CO 2 H
esterase
or
OCOCH 3
Figure A3.1
OH
OCOCH 3
CO 2 H CO 2 H CO 2 R
CO 2 R
R'COCl
NaOH/H 2 O
ROH
H 2 SO 4
R'COCl
pyridine
OCOR' OH OH
(3)
(1)
(2)
OCOR'
(4)
Figure A3.2
275