- The nature of the isoprenoid pathway and diversity of biologically
significant products suggests that the pathway may be highly regulated,
particularly under stress conditions, where the availability of high
energy intermediates is limited. It was postulated that germinating
seeds suffer from a limited supply of available energy and must therefore
conserve energy wherever possible. This assumption should be
reflected through enzymic changes in specific isoprenoid enzymes of
the pathway, correlated with the developmental requirements for
growth and differentiation.
An in vivo analysis of nonsaponifiable products formed from
mevalonate-2-¹⁴C versus germination in Pisum sativum confirmed
that β-amyrin (triterpene) and β-sitosterol (sterol) are sequentially
synthesized at different rates, depending upon the germination stage.
Kinetic transitions between squalene, squalene-triterpene, and
squalene-triterpene/squalene-sterol formation are discussed in relation
to the metabolic steps required for either triterpene or sterol
biosynthesis. Metabolically, either product must arise from 2, 3-oxidosqualene. Changes in triterpene and sterol formation as a function
of germination, however, suggest that the enzymes responsible
for such synthesis do not compete for oxidosqualene, but for squalene.
A regulatory model based on these observations is proposed. Squalene
epoxidase is postulated to combine with specific cyclases as an enzyme
aggregate. As an aggregate, the enzyme complex competes for
squalene to make the appropriate cyclized product. Regulation and
selection of product formation is controlled through association and
dissociation of the aggregate.
Methods for the biosynthesis and isolation of phosphomevalonate-2-¹⁴C, isopentenyl-4-¹⁴C pyrophosphate and farnesyl-4, 8, 12-¹⁴C pyrophosphate from mevalonate-2-¹⁴C in yeast autolysates, prepared
from commercial dry baker's yeast, are described in detail. Comparisons
between various biosynthetic methods have been made.
Methods of identification, isolation technics and criteria of purity
are also discussed; those methods most suitable for the isolation
and identification of each isoprenoid intermediate have been emphasized.
Individual assays of mevalonic kinase, phosphomevalonic
kinase, pyrophosphomevalonic decarboxylase, isopentenyl pyrophosphate isomerase, isopentenyl pyrophosphate/dimethylallyl
pyrophosphate prenyltransferase and squalene synthetase in 40,000 g
supernatants of Pisum sativum, prepared from germinating seeds at
selected intervals after the onset of imbibition, suggest that the enzymes
are regulated in such a manner as to always favor isopentenyl
pyrophosphate and dimethylallyl pyrophosphate formation. The enzymes
are activated in such proportions as to favor nonequilibrium
conditions between mevalonic acid and isopentenyl pyrophosphate, and
equilibrium conditions between isopentenyl pyrophosphate and dimethylallyl
pyrophosphate, thereby insuring the most efficient synthesis of
higher isoprenoid products at all times. A reversal of carbon flow
from dimethylallyl pyrophosphate to isopentenyl pyrophosphate
appears highly probable as a means of maintaining a sufficient supply
of isopentenyl pyrophosphate to meet the new biosynthetic requirements
induced by development. Dimethylallyl pyrophosphate may
serve as a storage pool, to meet biosynthetic demands in times of
stress, where the endogenous supply of mevalonic acid is limited.
Isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase
was assayed indirectly in 40,000 g supernatants with
mevalonate-2-¹⁴C and more directly with isopentenyl-4-¹⁴C pyrophosphate.
Under identical conditions in the preparation of 40,000 g
supernatants, the former assay yielded an estimated activity some
15-20 fold greater than by isopentenyl-4-¹⁴C pyrophosphate. These
results suggest that the prenyltransferase is allosterically activated
by one, or more, of the first three isoprenoid intermediates (i.e.,
mevalonate, phosphomevalonate, and/or pyrophosphomevalonate).
Indirect evidence was also obtained for the presence of two
distinct isopentenyl pyrophosphate/dimethylallyl pyorphosphate prenyltransferases, tentatively identified as enzymes I and II. Detection
of prenyltransferase I is independent of sucrose, while detection
of prenyltransferase II is highly dependent upon the inclusion of
sucrose (0.45 M) in the homogenizing buffer. The two prenyltransferases
are activated sequentially during the course of germination.
Prenyltransferase I is postulated to be associated with the cytoplasm,
while prenyltransferase II may be associated with an organelle, possibly
with immature chloroplasts (protoplasts) or mitochondria. Enzymic
changes in the activities of prenyltransferases I and II appear
closely correlated with developmental changes in the seed initiated
with the breaking of dormancy.
A thermolabile, large molecular weight factor has been tentatively
identified as a specific inhibitor of isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. This factor is temporal,
appearing at later stages of germination. The action of this
factor as a means of diverting the flow of carbon between cytoplasmic
and organelle associated pathways is discussed. Suppression of
prenyltransferase I activity by this factor, coincident with the rise in prenyltransferase II activity, may suggest that compartmentalized
isoprenoid pathways are not completely autonomous, but are suppressed
or activated to meet the needs of their respective environments,
thereby conserving considerable energy and substrate.