2. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the precursor for cholesterol synthesis. HMG-CoA is also an intermediate on the pathway for synthesis of ketone bodies from acetyl-CoA. The enzymes for ketone body production are located in the mitochondrial matrix. HMG-CoA destined for cholesterol synthesis is made by equivalent, but different, enzymes in the cytosol.

3.  HMG-CoA is formed by condensation of acetyl-CoA & acetoacetyl-CoA, catalyzed by HMG-CoA Synthase.  HMG-CoA Reductase catalyzes production of mevalonate from HMG-CoA.

4. The carboxyl of HMG that is in ester linkage to the CoA thiol is reduced to an aldehyde, and then to an alcohol. NADPH serves as reductant in the 2-step reaction. Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA.

5. HMG-CoA Reductase is an integral protein of endoplasmic reticulum membranes. The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme. The HMG-CoA Reductase reaction, in which mevalonate is formed from HMG-CoA, is rate- limiting for cholesterol synthesis. This enzyme is highly regulated and the target of pharmaceutical intervention.

6. Mevalonate is phosphorylated by 2 sequential P i transfers from ATP, yielding the pyrophosphate derivative. ATP-dependent decarboxylation, with dehydration, yields isopentenyl pyrophosphate.

7. Isopentenyl pyrophosphate is the first of several compounds in the pathway that are referred to as isoprenoids, by reference to the compound isoprene.

8. Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate & dimethylallyl pyrophosphate. Mechanism: protonation followed by deprotonation.

9. Prenyl Transferase catalyzes head-to-tail condensations:  Dimethylallyl pyrophosphate & isopentenyl pyrophosphate react to form geranyl pyrophosphate.  Condensation with another isopentenyl pyrophosphate yields farnesyl pyrophosphate.  Each condensation reaction is thought to involve a reactive carbocation formed as PP i is eliminated. Condensation Reactions

10. Each condensation involves a carbocation formed as PP i is eliminated.

11. Squalene Synthase: Head-to-head condensation of 2 farnesyl pyrophosphate, with reduction by NADPH, yields squalene.

12. Squaline epoxidase catalyzes conversion of squalene to 2,3-oxidosqualene. This mixed function oxidation requires NADPH as reductant & O 2 as oxidant. One O atom is incorporated into substrate (as the epoxide) & the other O is reduced to water.

13. Structural studies of a related bacterial enzyme have confirmed that the substrate binds at the active site in a conformation that permits cyclization with only modest changes in position as the reaction proceeds. The product is the sterol lanosterol. Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization.

14. Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes in ER membranes. Additional modifications yield the various steroid hormones or vitamin D. Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P 450 enzyme superfamily.