Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 134

4.2.4.3 Effect of Methyl Alcohol and Other Aliphatic Alcohols
on the Biosynthetic Activity of the Crude Homogenate

In order to study the alcohol specificity in the crude homogenate system, methanol
was replaced in the reaction mixture by a short chain primary alcohol (ethanol) a
secondary alcohol (isopropanol) or a tertiary alcohol (t-butanol). In all cases these
alcohols were unable to substitute for methanol (Rebeiz and Castelfranco 1971a ).
The effect of higher concentrations of methanol on the biosynthetic activity of the
system was subsequently investigated. It appeared that higher concentrations of
methanol were inhibitory.
To determine whether methanol acted as a catalyst or a substrate, the system was
incubated with 14 C-methanol. Mpe as well as Pchlidee and Pchlide ester eluted
from Silica Gel H were both labeled (Rebeiz and Castelfranco 1971a ). Although
these compounds were not purified to constant specific radioactivity, they remained
radioactive after elution from Silica Gel H and rechromatography on paper in
2,6-lutidine:0.1 n NH4OH (5:3.5 v/v) or acetone:petroleum ether:acetic acid
(3:7:0.01 v/v). These results suggested a substrate role for methanol in this system.
The differential incorporation of 14 C-methanol into 14 C-Pchlide and 14 C-Pchlide
ester (Rebeiz and Castelfranco 1971a ) supported the hypothesis that these two
compounds were produced via two separate pathways from a common precursor
(Rebeiz et al. 1970 ). Alternatively, it was conjectured that a certain degree of
transesterification of the alcohol of 14 C-Pchlide ester with 14 C-methanol may
have taken place. The possibility that methanol might be preferentially incorporated
into the phytol of Pchlide ester would also explain the observed incorporation data.
However, we are not aware of any direct pathway leading from methanol to
polyisoprenoids. The lower incorporation of 14 C-methanol into 14 C-Mpe and
14 C-Pchlide as compared to the incorporation of 14 C-ALA was expected under
these experimental conditions. Indeed by assuming that methanol esterifies the
propionic acid residue at the seventh position of the tetrapyrrole macrocycle,
eight molecules of ALA would be incorporated into the macrocycle for every
molecule of methanol utilized. Moreover, the specific radioactivity of the

14 C-methanol was about 34 time lower than that of 14 C-ALA.

4.2.4.4 Effect of pH and Temperature on the Biosynthetic
Activity of the Crude Homogenate

The multienzyme system in the crude homogenate appeared to have a pH optimum
of about 7.7 for the biosynthesis of 14 C-Mpe, and 14 C-Pchl(ide) (Rebeiz and
Castelfranco 1971a ). Some of the initial experiments were carried out at pH 7.9 before
it was recognized that the system was more active and more reproducible at pH 7.7.
The effect of two temperatures on the biosynthetic activity of the system was
also investigated (Rebeiz and Castelfranco 1971a ). At 20

C more 14 C-Mpe

accumulated than at 28 C whereas the incorporations into

14 C-Pchlide and

14 C-Pchlide ester were slightly depressed.