Cholesterol biosynthesis, site

Scheme 1. Sites of inhibition of cholesterol biosynthesis by Mevacor and zaragozic acid A.

Partial summary of lipoprotein metabolism in humans. I to VII are sites of action of hypolipidemic drugs. I, stimulation of bile acid and/or cholesterol fecal excretion II, stimulation of lipoprotein lipase activity III, inhibition of VLDL production and secretion IV, inhibition of cholesterol biosynthesis V, stimulation of cholesterol secretion into bile fluid VI, stimulation of cholesterol conversion to bile acids VII, increased plasma clearance of LDL due either to increased LDL receptor activity or altered lipoprotein composition. CHOL, cholesterol IDL, intermediate-density lipoprotein. 

Trifluoromethyl ketones and alcohol derivatives of squalene have been prepared in order to inhibit squalene epoxycyclase. This important enzyme regulates the biosynthesis of cholesterol. It bears a cysteine in its active site. Although these compounds have been shown to be good inhibitors, the involved mechanism is different from what was expected. Indeed, they do not inhibit squalene epoxycyclase, but they are substrates of this enzyme and are transformed into fluorohydroxysterols. The repression of the expression of HMG-CoA reductase is responsible for the observed inhibition of cholesterol biosynthesis. This repression comes from the back-regulation that is exerted by fluorohydroxysterols. Indeed, these compounds induce an important diminution of the cell activity of HMG-CoA reductase (Figure 7.66). °... 

In addition to their plasma membrane eukaryotic cells also contain internal membranes that define a variety of organelles (fig. 17.2). Each of these organelles is specialized for particular functions The nucleus synthesizes nucleic acids, mitochondria oxidize carbohydrates and lipids and make ATP, chloroplasts carry out photosynthesis, the endoplasmic reticulum and the Golgi apparatus synthesize and secrete proteins, and lysosomes digest proteins. Additional membranes divide mitochondria and chloroplasts into even finer, more specialized subcompartments. Like the plasma membrane, organellar membranes act as barriers to the leakage of proteins, metabolites, and ions they contain transport systems for import and export of materials, and they are the sites of enzymatic activities as diverse as cholesterol biosynthesis and oxidative phosphorylation. 

The synthesis of mevalonate is the committed step in cholesterol formation. The enzyme catalyzing this irreversible step, 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis, as will be discussed shortly. 

Although the mitochondria are the primary site of oxidation for dietary and storage fats, the peroxisomal oxidation pathway is responsible for the oxidation of very long-chain fatty acids, jS-methyl branched fatty acids, and bile acid precursors. The peroxisomal pathway also plays a role in the oxidation of dicarboxylic acids. In addition, it plays a role in isoprenoid biosynthesis and amino acid metabolism. Peroxisomes are also involved in bile acid biosynthesis, a part of plasmalogen synthesis and glyoxylate transamination. Furthermore, the literature indicates that peroxisomes participate in cholesterol biosynthesis, hydrogen peroxide-based cellular respiration, purine, fatty acid, long-chain... 

The major site of steroid synthesis in animals has been considered to be the liver, but recently (1975) this has been disputed and detailed studies now show that sterol synthesis in guinea pig occurs more readily in ileum and lung than in liver under a variety of conditions" " and a brief study indicates similar sites of biosynthesis in swine." All tissues of guinea pig studied had an active feedback system controlling cholesterol biosynthesis and the results of feeding cholesterol and cholestyramine" showed that both the former and possibly bile acids suppress cholesterol synthesis in the liver to a far lesser extent than in the small intestine. 

Cholesterol can be obtained from the diet or it can be synthesized de novo. An adult on a low-cholesterol diet typically synthesizes about 800 mg of cholesterol per day. The liver is the major site of cholesterol synthesis in mammals, although the intestine also forms significant amounts. The rale of cholesterol formation by these organs is highly responsive to the cellular level of cholesterol. This feedback regulation is mediated primarily by changes in the amount and activity of 3-hydroxy 3 methylglutaryl CoA reductase. As described earlier (p. 739), this enzyme catalyzes the formation of meval-onate, the committed step in cholesterol biosynthesis. HMG CoA reductase is controlled in multiple ways ... 

As will be described in this chapter, a highly specific system is present within the cell, i.e., sterol carrier proteins, which is capable of binding sterols (or squalene) and transporting them to specific enzyme sites involved in cholesterol biosynthesis and utilization. 

Fig. 8 is a schematic diagram of a cell which shows the known sites in which sterol carrier proteins are involved in cholesterol biosynthesis, utilization and intracellular transfer. SCP, participates in the conversion of squalene to lanosterol and SCP2 participates in the conversion of lanosterol to cholesterol, the conversion of cholesterol to cholesterol ester by ACAT, and probably also in the conversion of cholesterol to 7a-hydroxycholesterol. SCPj transfers cholesterol from cytoplasmic lipid inclusion droplets to mitochondria in the adrenal and SCPj also translocates cholesterol from the outer to the inner mitochondrial membrane. 

An elevated serum cholesterol level is an important risk factor in cardiac disease (and in hypertension), and thus a drug which could lower this level would be an important prophylactic against cardiovascular diseases in general. Humans synthesize about 50% of their cholesterol requirement, with the rest coming firom diet. A potential site for inhibition of cholesterol biosynthesis is at the rate-limiting step in the system, the reduction of hydroxymethyl-glutaryl... 

In animals, lovastatin is more efficiently extracted by the liver where it is converted to the active enzyme inhibitor. Accordingly, the systemic bioavailability of active inhibitors is less than 5% of an oral dose of lovastatin. The high hepatic extraction and low systemic availability are desirable features since the liver is the primary site of cholesterol biosynthesis. 

Much of the endogenous lipid that is eventually used by peripheral tissues is transported in the form of water-soluble ketone bodies, the two most important being jS-hydroxybutyrate and acetoacetate. The metabolic pathway of ketone body formation and its relationship to cholesterol biosynthesis is shown in Fig. 4.10. Four enzymes are Involved in the formation of ketone bodies, namely acetyl-CoA transferase (also known as thiolase), hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase), hydroxymethyl-glutaryl-CoA lyase (HMG-CoA lyase) and jS-hy-droxybutyrate dehydrogenase. Tbe last of these catalyses the interconversion of the two principal ketone bodies. All four enzymes are present in liver, the principal site of ketone body formation. Acyl-CoAs are unable to pass through the plasmalemma, and HMG-CoA lyase thus controls the release of ketone... 

Lovastatin, because a part of its structure resembles mevalonate ion, can apparently bind at the active site ot HMGA-CoA-reductase and act as a competitive inhibitor ot this enzyme and thereby reduce cholesterol biosynthesis. 

Reasoning that a compound which might inhibit cholesterol biosynthesis could be useful for treatment of hypercholesteremia and atherosclerosis, investigators synthesized and tested a number of compounds in vitro and in vivo. With the knowledge and techniques gained from the work on cholesterogenesis, it has been possible to assign the sites at which the various compounds inhibit biosynthesis. 

The formation of mevalonate is the rate-limiting step in cholesterol biosynthesis. Drugs that inhibit HMG-CoA reductase are termed statins and are one of the most widely used types of prescription drugs. All statins share a structural component that is similar to the 3-hydroxy-3-methylglutaryl portion of HMG-CoA reductase. AH statins differ from HMG-CoA in being more bulky and more hydrophobic [10]. Statins occupy the binding site of HMG-CoA thus blocking access of this substrate [11]. All statins share this common mode of action but differ in their overall structural, biochemical, thermodynamic, and pharmokinetic properties which influences their efficacy [12]. 

FIGURE 1.1 The metabolic conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate is a crucial step in the body s pathway for biosynthesizing cholesterol. An X-ray crystal structure ofthe active site in the HMG-CoA reductase enzyme that catalyzes the reaction is shown, along with a molecule ofatorvastatin (Lipitor) that is bound in the active site and stops the enzyme from functioning. With the enzyme thus inactivated, cholesterol biosynthesis is prevented. 

Fio. 2. Simplified scheme of cholesterol biosynthesis up to squalene formation. Dotted arrow refers to the site of feedback, control of cholesterogenesis. 

Fio. 3. Last steps of cholesterol biosynthesis. Dotted arrows refer to sterol intermediates with 30, 29, 28 and 27 carbon atoms with saturated or unsaturated side chain. (1) sites of action of A -reductase (2) sites of action of A -reductase. 

Bile acids and cholesterol are partially reabsorbed through the enterohepatic cycle. The formation of bile acids is homeostatically controlled by their concentrations in the portal vein and the specific site of this feed-back mechanism could well be at the 7 a-hydroxylation step of cholesterol Bile acids may also exert a direct control on cholesterol biosynthesis. 

While these enzymes are less sensitive to cholesterol repression than HMG-CoA reductase, they are at least potentially loci for an adaptive, secondary mechanism of regulation. These findings are too recent to allow any assessment or reassessment of the main control sites in cholesterol biosynthesis. What one would wish to know inter alia is whether cytoplasmic thiolase and HMG-CoA synthetase show a similar circadian pattern as HMG-CoA reductase and whether they respond in a like manner to the various changes in physiological state. 

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