Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

Enzymatic Carbon–Carbon Bond Formation 181
OPP OPP PPO −
PPO
H
−H +
geranyl PP
3S-linalyl PP
myrcene (9%)
−H + =
−H +
H
−H + −H +
(−)-β-pinene (21%)
H
1,2-shift
H
(−) limonene (8%)
1,2-shift
H
(−)-α-pinene (26%)
(−) camphene (28%)
Figure 7.32 Mechanism for (
)-a-pinene synthase.
elimination from the monocyclic carbocation, whilst camphene is formed
by two consecutive 1,2-alkyl migrations, followed by elimination.
Most enzymes are speciWc for the production of a single isomeric product,
so in this case how can we be sure that all of these products are from one
enzyme Apart from demonstrating that there is only one homogeneous protein
present, a chemical test can be made. If there is a common precursor to two or
more products, then by introducing deuterium atoms at speciWc points in the
substrate, the ratio of products should be aVected by the presence of a deuterium
isotope eVect, which should favour one pathway over another. This test
was applied to the ( )-pinene synthase reaction by incubation of the speciWcally
deuterated substrate shown in Figure 7.33. At the point of divergence to a-
pinene or b-pinene the presence of a terminal 2CD 3 group would be expected
to favour formation of a-pinene. This was indeed observed: the proportion of
a-pinene in the product mixture rose from 26% to 38%, whilst the proportion
of b-pinene dropped from 21% to 13%. The proportion of myrcene product
also dropped signiWcantly from 9% to 4%, also consistent with an isotope eVect
operating on the allylic carbocation intermediate.
Farnesyl pyrophosphate is cyclised to form the large family of sesquiterpene
natural products by a further class of terpene cyclase enzymes, several of which
have been puriWed to homogeneity. Pentalenene synthase catalyses the cyclisation
of farnesyl pyrophosphate to give pentalenene, which is further processed
in Streptomyces UC5319 to give the pentalenolactone family of antibiotics, as
shown in Figure 7.34. The enzyme is a 41-kDa protein with a K M of 0.3 m for