The biosynthesis of cholesterol is well covered in standard biochemistry textbooks and only the more important features will be outlined here. However, it should be added that a great deal of work has gone into solving the pathway of cholesterol formation with those concerned receiving no less than 13 Nobel prizes between them!
The precursor pool in mammalian cells is the cytosolic acetyl-CoA. This acetyl-CoA may be derived, for example, from (3-oxidation of fatty acids by mitochondria or microbodies (Section 2.3.1). The acetyl-CoA pool is in rapid equilibrium with intracellular and extra-cellular acetate, which allows radiolabelled acetate to be used conveniently to measure cholesterol synthesis in tissues.
The first two steps involve condensation reactions catalysed by a thiolase and hydroxy-methylglutaryl-CoA (HMG-CoA) synthetase. Both enzymes are soluble and the first reaction is driven to completion by rapid removal of acetoacetyl-CoA by the second step (Fig. 7.18).
HMG-CoA synthetase has been studied in considerable detail and the reaction mechanism defined. It shows a very high degree of specificity with regard to the stereochemistry of the aceto-acetyl-CoA substrate and the condensation proceeds by inversion of the configuration of the hydrogen atoms of acetyl-CoA. In addition to cytosolic HMG-CoA synthase, a second synthase is found in mitochondria. Not only has this been shown to be a different protein, the HMG it forms has a different function. HMG in the cytosol is destined for mevalonate formation, whereas in mitochondria it is broken down by HMG-CoA lyase to yield acetyl-CoA and acetoacetate (Section 22.214.171.124). The cytosolic HMG-CoA synthase seems to be one of the pathway enzymes important for controlling cholesterol synthesis and its activity is changed by transcriptional modulation.
The next reaction, which results in mevalonate production, is catalysed by the membrane-localized HMG-CoA reductase. It is a highly regulated enzyme with a short half-life (Ty2 about 3 h) and it is usually considered to catalyse the reaction with the most control over the rate of sterol synthesis. Even in a normal diurnal cycle, its activity will vary about tenfold.
HMG-CoA reductase activity is regulated at the transcriptional level and by post-transcriptional methods. When sterols are added to animal diets there is a decline in the mRNA levels for HMG-CoA reductase (together with HMG-CoA synthase, farnesyl diphosphate synthase and the LDL-recep-tor (Section 5.3.1). These four mRNAs increase when cells are deprived of sterols. Brown and Goldstein showed that the 3'-flanking region of the gene for HMG-CoA reductase (and the LDL-receptor) contained one to three copies of a nucleotide sequence called the sterol regulatory element I (SRE-I). A transcription factor, sterol regulatory element binding protein I (SREBP-I) specifically binds to SRE-I, which is within the promoter for HMG-CoA reductase (and the LDL-receptor). The transcription factor, SREBP-I, is synthesized as a 125 kDa precursor bound to the endoplasmic reticulum. In cells deprived of cholesterol, the factor is cleaved and a 68 kDa N-terminal fragment is released, which is targeted to the nucleus, where it binds to SRE-I and promotes expression of HMG-CoA reductase.
HMG-CoA reductase expression is also regulated by changes in mRNA translation and stability and by protein turnover. The degradation of HMG-CoA reductase protein in the endoplasmic reticulum is regulated through the eight trans-membrane domains, perhaps by farnesol or its diphosphate derivative or via oxysterols.
HMG-CoA reductase is also regulated by a reversible phosphorylation/dephosphorylation cycle (Fig. 7.19). The phosphorylated reductase is inactive and the amounts of the phosphorylated enzyme can be shown to be increased when its activity is decreased by mevalonate or glucagon.
The protein kinase, which inactivates HMG-CoA reductase, is itself subject to phosphorylation. Interestingly, both ATP and ADP are needed. Apparently ADP binds to a different site on the reductase kinase and acts as an allosteric effector. The phosphatases, which activate HMG-CoA reductase, are highly sensitive to NaF. The kinase and phosphatases are each present in the cytosol as well as endoplasmic reticulum. Recently the protein kinase, which phosphorylates HMG-CoA reductase, has been identified as an AMP-activated protein kinase. Interestingly, this kinase also phosphorylates (and thus regulates) acetyl-CoA carboxylase (Section 126.96.36.199).
Because of the involvement of cholesterol in the aetiology of arterio-vascular disease (Section 5.4) considerable efforts have been made to develop suitable pharmaceutical agents able to reduce its formation. Two interesting compounds that inhibit HMG-CoA reductase are natural antibiotics isolated from the moulds Penicillium spp. and Aspergillus terreus and named compactin and mevinolin, respectively. These compounds are competitive inhibitors of HMG-CoA reductase with a Ki for the enzyme of about 10-9M compared to 10-SM for HMG-CoA. Surprisingly, they increase mRNA and protein levels for the enzyme, but this is consistent with a downstream metabolite of mevalonate inhibiting transcriptions and HMG-CoA reductase turnover (as discussed above). Nevertheless, these fungal metabolites rapidly enter cells and inhibit cholesterol biosynthesis (Section 5.3.1).
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