Hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase) catalyzes the
formation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA. F-244 (1), a naturally
occurring J3-lactone isolated from Fusarium sp. ATCC 20788 and other species, is known
to be a potent and specific inhibitor ofHMG-CoA synthase isolated from rat liver.
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This thesis describes the 48 fold purification of HMG-CoA synthase from
bakers yeast in a three step procedure involving ethanol fractionation followed by
ammonium sulfate precipitation and then hydroxylapatite chromatography. This
procedure was found to be reproducible and yields a preparation of specific activity 0.14
units (j...tmolfmin)/mg in an overall yield of 8%.
In our study, F-244 was found to be a potent irreversible inhibitor of HMGCoA
synthase isolated from bakers yeast, with an IC50 value of 0.009 j...tM. This value is
almost identical to the inhibitory activity of F-244 on rat liver HMG-CoA synthase that
has been reported in the literature.
Tritium labeled F-244 was prepared, for the first time, by feeding methyl-[3H]methionine
to cultures of Fusarium sp. The [15, 16, 17, 18-3H] F-244 isolated had a
specific activity of 1.3 x 106 DPM/mg. This tritiated F-244 was then used as an affinity.label for HMG-CoA synthase. Attempts to isolate the enzyme-inhibitor complex were
unsuccessful due to the low level of radioactivity associated with the tritiated F-244.
HMG-CoA synthase was also shown to be inhibited in a time-dependent
irreversible manner by {±)-(3-butyrolactone (2). The rate of inactivation (~) was found to
be 0.4697 s-1 and the inhibition constant (K1) was found to be 9 mM. The inactivation was
found to be irreversible over several hours. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22772 |
| Date | 08 1900 |
| Creators | Bell , Karen Lesley |
| Contributors | Harrison, Paul, Chemistry |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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