Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Enzymatic Transformations of Amino Acids 213
2− O 3 PO
B −
1 B 2 H B 1 B 2
B 1 H B −
2
R
H
H H
CO −
2 R CO −
CO −
2 R 2
H
NH +
NH + NH +
OH
2− OH
O 3 PO
2− OH
O 3 PO
+
N
+
N
H
N
H H
aldimine intermediate quinonoid intermediate
Figure 9.3 Mechanism for PLP-dependent racemases (two-base mechanism illustrated).
R
CO −
2
H
NH +
3
results in inversion of conWguration at the a-centre, as shown in Figure 9.3.
Detachment of the product from the coenzyme is carried out by attack of the
active site lysine residue.
In some racemases reprotonation is carried out by a second active site
residue (the ‘two-base’ mechanism). In other cases deprotonation and reprotonation
are carried out by a single active site base that is able to access both faces
of the ketimine adduct. The latter ‘one-base’ mechanism can be demonstrated
in a single turnover experiment by incubating a 2- 2 H-l-amino acid substrate
with a stoichiometric amount of enzyme. Isolation of d-amino acid product
containing deuterium at the a-position implies intramolecular atom transfer by
a single active site base.
One important example of a PLP-dependent racemase is alanine racemase,
which is used by bacteria to produce d-alanine. d-alanine is then incorporated
into the peptidoglycan layer of bacterial cell walls in the form of a d-Ala-d-Ala
dipeptide. Inhibition of alanine racemase is, therefore, lethal to bacteria, since
without peptidoglycan the cell walls are too weak to withstand the high osmotic
pressure, and the bacteria lyse. One inhibitor of alanine racemase that has
antibacterial properties is b-chloro-d-alanine, which inhibits the enzyme via
an interesting mechanism shown in Figure 9.4. b-Chloro-d-alanine is accepted
as a substrate by the enzyme, which proceeds to bind the inhibitor covalently
to its PLP cofactor. However, once in 800 turnovers deprotonation at the
a-position is followed by loss of chloride, generating a PLP-bound enamine
intermediate. This is detached from the PLP cofactor by attack of the lysine
e-amino group; however, the liberated free enamine reacts with the carbon
centre of the PLP-enzyme imine, generating an irreversibly inactivated species.
Further examples of such mechanism-based inhibitors will be given in the
Problems section. Note also that there is a family of cofactor-independent
racemase/epimerase enzymes which will be discussed in Section 10.2.
Amino acid decarboxylases proceed from the PLP-amino acid adduct
shown in Figure 9.2, this time using the PLP structure as an electron sink for
decarboxylation of this adduct. Reprotonation at (what was) the a-position,
followed by detachment of the product from the PLP cofactor, generates the