Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

76 Chapter 4
Table 4.3 Group speciWc reagents for active site amino acid modiWcation.
Amino acid Group modiWed Reagent ModiWcation reaction
Cysteine S Iodoacetate, iodoacetamide Alkylation
DTNB
p-Hydroxymercuribenzoate
Disulphide formation
Metal complexation
Histidine N Diethyl pyrocarbonate Acylation
Lysine N Succinic anhydride Acylation
Arginine N Phenylglyoxal Heterocycle formation
Asp, Glu O EDC (water-soluble carbodi-imide) þ amine Amide formation
Tyrosine O Tetranitromethane Nitration
Tryptophan Indole N-bromosuccinimide Oxidation
DTNB, 5,5 0 -dithiobis-(2-nitrobenzoic acid); EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.
active site residue. The enzyme is then irreversibly inactivated, since the active
site is blocked. The advantage of this method over the group speciWc reagents
mentioned above is that aYnity labelling is more selective in its site of action,
due to the precise positioning of the substrate analogue at the enzyme active
site. One example of this method is the inactivation of the serine proteases
by chloromethyl ketone substrate analogues, which will be described in
Section 5.2.
If a successful irreversible inhibitor is found, the site of action of the aYnity
label can be determined using a radiolabelled inhibitor as shown in Figure 4.29.
The enzyme inactivated with labelled inhibitor contains a modiWed active site
E + I*
irreversible
inhibition
E-I*
protease
I*
HPLC
purification
protein sequence
peptide sequence
..K F D A L H S M Y V..
D A L * S M Y
peptide
sequencing
Figure 4.29 IdentiWcation of an active site residue by peptide mapping. HPLC, high-performance
liquid chromatography.
I*