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182 Chapter 7
CH 3
*
OPP
(−)pinene
synthase
−H *+
Mg 2+ H
* H 3 C
CH*
2
β-pinene H* = H 21%
−H +
H* = D 13%
Figure 7.33 Kinetic isotope eVect in the (
CH*
3
α-pinene H* = H 26%
H* = D 38%
)-pinene synthase reaction.
farnesyl pyrophosphate and a k cat of 0:3s 1 , and an absolute requirement for
Mg 2þ ions. A series of isotopic labelling experiments have been carried out to
support the mechanism of cyclisation shown in Figure 7.34. Cyclisation of
farnesyl pyrophosphate is proposed to form an 11-membered intermediate,
humulene, which is followed by a Wve-membered ring closure to form a bicyclic
tertiary carbocation. 1,2-Hydride migration followed by a further Wvemembered
ring closure gives a tricyclic carbocation, which upon elimination
gives pentalenene.
Examination of the inferred amino acid sequence of pentalenene synthase
revealed an amino acid sequence motif Asp–Asp–X–X–Asp (DDXXD) found
in other pyrophosphate-utilising enzymes. It is believed that the Asp–Asp pair
are involved in chelating the essential Mg 2þ ion, which in turn chelates the
pyrophosphate ion as shown in Figure 7.35. An indication of the tight binding
of pyrophosphate is that this product inhibits the enzymatic reaction strongly
(K i 3 m).
H
− BEnz
H
BEnz
PPO
humulene
H
H
H
CO 2 H
H
H
H
− BEnz
O O
O
pentalenene
pentalenolactone
Figure 7.34 Mechanism for pentalenene synthase.
H