Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

200 Chapter 8
*H
R
H
H
CO 2
−
NH 3
+
R = Ph
R = imidazole
R = CO 2 H
R
CO 2
−
+
NH 4
+
phenylalanine ammonia lyase
histidine ammonia lyase
aspartase
Figure 8.11 Reactions catalysed by some ammonia lyases.
broken lies adjacent to an activating group in the b-position of the amino acid
side chain.
The histidine and phenylalanine ammonia lyases utilise a novel mechanism
to assist the leaving group properties of ammonia, which would normally be a
poor leaving group. These enzymes are readily inactivated by treatment with
nucleophilic reagents, implying that there is an electrophilic group present at the
enzyme active site. Examination of puriWed enzyme revealed that the electrophilic
cofactor has a characteristic ultraviolet (UV) absorption at 340 nm.
Inactivation of phenylalanine ammonia lyase with NaB 3 H 4 followed
by acidic hydrolysis of the protein gave 3 H-alanine, suggesting that the electrophilic
group might be a dehydroalanine amino acid residue, formed by dehydration
of a serine residue. However, determination of the X-ray crystal structure
of Pseudomonas putida histidine ammonia lyase in 1999 revealed that the
electrophilic prosthetic group is 3,5-dihydro-5-methylene-imidazolone, formed
by dehydration of Ser-143, and cyclisation of the amide nitrogen of Gly-144
with the amide carbonyl of Ala-142, as shown in Figure 8.12. Substitution of
Ser-143 for threonine using site-directed mutagenesis gives an inactive enzyme;
however, a Cys-143 mutant enzyme is able to form the dehydroalanine cofactor
by elimination of hydrogen sulphide.
Two possible mechanisms for the phenylalanine ammonia lyase reaction are
illustrated in Figure 8.13. Attack of the a-amino group of the substrate upon
the methylene group of the cofactor (path A), followed by 1,3-prototropic shift,
generates a secondary amine intermediate, in which the amine is activated as an
enamide leaving group. Anti-elimination of this intermediate leaves the amine
covalently attached to the cofactor. Prototropic shift, followed by elimination
of ammonia, regenerates the methylene–imidazolone cofactor.
NH
142 O
144
HN
HN 143
O
H
N
O
NH
N
N
O
H
N
O
OH
Figure 8.12 Methylene-imidazolone cofactor.