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Enzymatic Addition/Elimination Reactions 205
Intermediate
CO 2
−
Phosphonate analogue
CO 2
−
2− O 3 PO
OH
O
CH 3
CO
−
2
OPO
2−
3
2− O 3 PO
CO
−
2
PO
2−
3
resonance (NMR) spectroscopy, verifying the structure of the tetrahedral intermediate
previously suspected. This intermediate was subsequently observed
transiently at the active site of the enzyme by NMR spectroscopy. Synthetic
phosphonate analogues of the tetrahedral intermediate have been synthesised
and were found to be potent inhibitors of the enzyme, as shown in Figure 8.19.
The X-ray crystal structure of EPSP synthase is shown in Figure 8.20.
Examination of the structure reveals that the enzyme contains two domains
connected by a Xexible hinge, suggesting that there is a conformational change
of the protein occurring during catalytic turnover.
EPSP synthase is of signiWcant commercial interest, since it is the target for
a herbicide called glyphosate, or phosphonomethyl-glycine. Inhibition of the
shikimate pathway in plants is catastrophic, since the plant can no longer
synthesise the aromatic amino acids required for metabolism and for the construction
of the structural polymer lignin. However, glyphosate is non-toxic to
OH
Figure 8.19 Tetrahedral intermediate in the EPSP synthase reaction.
O
CH 3
Figure 8.20 Structure of EPSP synthase (PDB Wle 1G6S), complexed with glyphosate (shown in red)
and shikimate-3-phosphate (shown in black). Situated beneath the active site is a ‘hinge’ region,
which mediates a protein conformational change.