METABOLISM

The metabolic processes and reactions that occur in our cells are the essence of life. They are also very intricate and detailed. The following is meant to be only an overview of some of the important reactions. For some questions regarding what is on this page, click here.

Glycolysis

Glycolysis is the anaerobic conversion of glucose to pyruvate with a net gain of 2 molecules of ATP for every glucose molecule that is broken down. It is a prime example of the regulation of enzymes.

One main rate limiting reaction that occurs early in glycolysisis the phosphorylation of fructose 6 phosphate (F-6-P) to fructose 1,6 bisphosphate (F-1,k-BP), catalyzed by  phosphofructokinase (PFK1). The enzyme, PFK1 is allosterically activated by fructose 2, 6 bisphosphate (F-2,6-BP).  F-2,6-BP is also a product of the phosphorylation of F-6-F but in a reaction that is catalyzed by phosphofructokinase2 (PFK2).

Another rate limiting reaction that occurs at the end of glycolysis is the transfer of a phosphate group from phosphoenolypyruvate to ADP to yield Pyruvate and ATP. This reaction is catalysed by pyruvate kinase which can exist in 1) a less activated phosphorylated form or 2) in a more active  dephosphorylated form. Pyruvate kinase is slowed down by the phosporylating hormone, glycogen, and activated by the dephosphyrlating hormone, insulin.

In certain animal tissues like muscle when inadequate oxygen is present, pyruvate is converted into lactate. In this process, the NADH produced by glycolysis gives up its electrons and is converted into NAD⁺.

Gluconeogenesis

Gluconeogenesis is the biosynthesis of glucose which occurs predominantly in the liver by a process that is essentially a reversal of glycolysis. Lactate which is producing during anaerobic glycolysis in muscle is a predominate source of carbon atoms for glucose synthesis by gluconeogenesis. The lactate that is produced in the muscle is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned by the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle.

Because gluconeogenesis is essentially a reversal of glycolysis, the positive and negative effectors of glycolysis have reverse roles in gluconeogenesis. For example, unlike the first rate limiting reaction in glycolysis where PFK1 is stimulated by 2,6,-bisphosphate, the enzyme, fructose 1,6, bisphosphatase (F-6,6-BPase), which catalyzes the conversion of 1,6,-bisphoshate to fructose 6-phosphate in gluconeogenesis is decreased by high levels of 2,6-biosphosphate. Conversely, the presence of ATP which inhibits the glycolysis forward reaction stimulates fructose 1,6, bisphosphatase and the production of glucose.

The rate limiting reaction of the conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis is catalyzed by the enzyme, phosphoenolpyruvate carboxylkinase (PEPCK). In contrast to the activity of pyruvate kinase in glycolysis, PEPCK is induced in response to glucagon. The mechanism of action here is rather unique in that glucagon promotes transcription of a gene that encodes PEPCK through a kinase mediated signalling cascade (cAMP). 

When thinking about the regulation of key enzymes in glycolysis and gluconeogenesis, the effectors make inherent sense. When blood glucose is high, insulin and the rate of glycolysis is up. When blood glucose is low, most cells go into an energy saving mode; glucagon and the rate of gluconeogenesis are up, PFK1 and PFK2 are off.

Citric Acid Cycle:

The citric acid cycle is also known as the tricarboxylic acid cycle or the Krebs cycle. Each turn of the cytric acid cycle produces 3 molecules of NADH, 1 molecule of FADH₂ and 1 molecule of GTP. The energy that is stored in the readily transferred high energy electrons of NADH and FADH₂ is subsequently utilized for ATP production through a process called "oxidative phosphorylation" which is discussed below.

Before the citric acid cycle can start, both acetyl CoA and oxaloacetate are required. The 2 main precursors for precursors for acetyl CoA are pyruvate and fatty acids. Both of these fuel molecules are transported across the inner mitochondrial membrane and then converted to acetyl CoA by enzymes located in the mitochondrial matrix. In the case of pyruvate, acetyl CoA formation is catalyzed by a complex of 3 enzymes called the pyruvate dehydrogenase complex. Pyruvate is also a precursor for the formation of oxaloacetate through a reaction catalyzed by pyruvate carboxylase.

The citric acid starts off with the joining of the 4 C unit, oxaloacetate with the 2 C unit, acetyl CoA in a reaction catalyzed by citrate synthase to form citrate which is in turn isomerized into isocitrate in a reaction catalyzed by aconitase.

In the 1st of the 4 oxidation steps in the cycle, isocitrate + NAD is converted into alpha-ketoglutarate + NADH in a reaction catalyzed by iscitrate dehydrogenase.

The second oxidation step comes when alpha ketoglutarate + NAD is converted into succinyl CoA + NADH in a reaction that is catalyzed by the alpha ketoglutarate dehydrogenase complex. This complex closely resembles the large enzyme complex, pyruvate dehydrogenase above which converted pyruvate to acetyl CoA.

Next, succinyl CoA + GDP is converted into succinate + GTP in a reaction catalyzed by succinyl CoA synthetase.

In the third oxidation step in the cycle, FAD removes 2 H atoms from succinate to form fumarate in a reaction catalyzed by succinate dehydrogenase. Fumarate is then converted into malate in a reaction catalyzed by fumarase.

In the last of 4 oxidation steps, malate + NAD yields oxaloacetate + NADH in a reaciton catalyzed by malate dehydrogenase. The regeneration of oxaloactetate allows the cycle to continue once again.

The citric acid cycle is not only important because is gnerates high energy electrons in the form of NADHwhich are passed to a membrane bound electron transport chain in oxydative phosphorylation which is discussed next, but also because many of the products above are intermediates for biosyntheses in other cycles as shown below.

Oxidative Phosphorylation:

Oxidative phosphorylation produces about 2.5 molecules of ATP from the pair of electrons donated by NADH and 1.5 ATP molecules per electron pair from from FADH₂. Without mitochondria where oxidative phosphorylation occurs, eucaryotes would be dependent on the relatively inefficient process of glycolysis for all of their ATP production.

Oxidative phosphorylation occurs in the inner mitochondrial membrane. NADH which is formed by glycolysis in the cytosol presents a problem since NADH can not diffuse across the inner mitochondrial membrane. The NADH that is generated during glycolysis is brought into the mitochondrial membrane using what is called the glycerol-3-phosphate shuttle. Another way that electrons from the cytosol enter the mitochondrial membrane is by the malate aspartate shuttle.

In oxidative phosphorylation, electron motive force is converted into proton motive force and then into phosphoryl potential. As electrons from reduced substrates flow through various complexes, protons are translocated across the inner mitochondrial membrane from the matrix to the intermembrane space. This creates a proton (H+) gradient which is negative on the matrix side and + on the cytosolic side of the inner mitochondrial membrane. As protons reenter the mitochondrial matrix due to this gradient, their reentry is coupled to the conversion of ADP and P_i to ATP. This conversion takes place in a complex known as the ATP synthase which is composed of 3 subunits. One unit called F- spans the inner membrane and serves as the proton channel of the complex. Another subunit, F1 contains the catalytic sties for ATP synthesis.

ATP and ADP do not freely diffuse across the inner membrane but must be transported using an ATP-ADP translocase. When this transport protein bind ADP, there is an eversion of the protein whereby ADP is transported in from the cytosolic side. Once inside the matrix, ADP is released. ATP then bind causing another eversion of the protein which transports ATP out to the cytosolic side.

Certain drugs can uncouple oxidative phosphorylation such as DNP and thermogenin.

Cholesterol and Lipoproteins

Cholesterol is an essential component of membranes. Elevated levels of cholesterol can also be very dangerous leading to such diseases as atherosclerosis. Statins (ex. fluvastatin) are commonly used to lower cholesterol levels.

The rate limiting reaction in the  biosynthesis of cholesterol is the reduction (NADPH to NADP+) of 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) to Mevalonate, catalyzed by the enzyme HMG-CoA Reductase. HMG-Co is a product which can also be cleaved to form the ketone body  acetoacetate (see below). But that reaction occurs in the mitochondria whereas the reduction of HMG-CoA to mevalonate occurs in the cytosol. Other reactions after the product of mevalonate leading to the synthesis of cholesterol do occur in the mitochondria. The enzyme HMG-CoA reductase spans the ER membrane 3 times. Its active site, however, is actually on the cytoplasmic side of the ER membrane. HMG-CoA reductase is regulated using transcription mechanisms by 1. insuline (+) [after a meal, insulin levels increase which turns on cholesterol synthesis and in turn bile acid synthesis; 2. glucagon (-); 3. thyroid hormone (+), 4. statins (+) and bile acids (-) [if have enough bile acids no sense in making more cholesterol]. HMG-CoA reductase is regulated using translation mechanisms by 1. cholesterol which is the end product of the pathway.

Most of the cholesterol in our bodies is esterified using 1 of 2 different reactions. The 1. LCAT (lecithin cholesterol acyltransferase) reaction of 2. the ACAT (acyl CoA cholesterol acyltransferase) reaction.

Elimination of cholesterol from the bile requires bile acids. The rate limiting reaction in the biosynthesis of bile acids is the reduction (NADPH to NADP+) of cholesterol to 7α-hydroxycholesterol by the enzyme cholesterol 7-α-hydroxylase. This rate limiting step occurs in the mitochondria of the liver. The next steps in the biosynthesis occur in peroxisomes of the liver and in the final steps, bile acids are conjugated with either glycine or taurine in the cytosol. The enzyme cholesterol 7-α-hydroxylase is regulated by 1. thyroid hormone (+) 2. cholesterol (+) and 3. bile acids (-). Bile acids are returned to the liver via the enterohepatic circulation.

Cholesterol and triacylglycerols are transproted in the body in the form of lipoproteins. Cholesterol esters or triacylglycerols are contained on the inside of such lipoproteins (no charge) and polar groups form the exterior of the lipoprotein. There are several types of lipoproteins, each of which has a particular apolipoprotein surface which serves as the ligand for a specific lipoprotein receptor.

1. chylomicrons are the lowest density type of lipoprotein. Chylomicrons are produced in the intestinal mucosal cell and have the apolipoprotein B-48 which forms an amphiphathic shell round the fat globule. The chylomicrons travel to the lining of blood vessels in tissues that use fatty acids as fuels where the cylomicrons are hydrozlymed by lipoprotein lipases. Lipoprotein lipases are activated by the apolipoprotein C-II. Chylomicron remants then travel to the liver.

2. Low density lipoproteins are divided into 3 groups (VLDL, IDL, and LDL) and all have the apolipoprotein B100 which is encoded by the same gene as the Apo B48 for chylomicrons but is larger because it has not been edited. VLDL (very low density lipoproteins) are formed in the liver (they are used to export the triacylglyerols in excess of the liver's needs) and then travel to the lining of blood vessels where like chylmicrons, they are hydrolyzed by lipases. The resulting remnants are rich in cholesterol ester called IDL (intermediate density lipoproteins). Half of the IDL are taken up by the liver whereas the other half is converted to LDL (low density lipoproteins)  which are the major carrier of cholesterol in the blood. LDL's travel to the lining of blood vessels where where the apoliprotein 100 binds to specific LDL receptors. LDLs are internalized by endocytosis to form a vesicle which then fuses with lysosomes which have enzymes that hydrolyze the protein component of the LDL. The LDL receptor is then free to retun to the PM and the cholesterol is free for use for membrane biosynthesis or can be reesterified for storage inside the cell by ACAT (acyl CoA: cholesterol acyltransferase) discussed above. Defects in LDL receptors leads to a very dangerous condition called familial hpercholesterolemia (FH) which results in the accumulation of LDL in thhe plasma which can lead to atherogenesis. Oxydation of LDL and uptake by scavenger receptors are key events in atherogenesis (damaging of the endothelium tissues). Homozygotes for this disease have amost no LDL receptors whereas heterozygotes for the defficient gene have half the normal number. HDL (high density lipoproteins) which are sometimes referred to as "good cholesterol" are involved in the reverse transport of cholesterol from the periopheral cells back to the liver cells. The cholesterol is transported out of the cells to HDL using ABC1 protein transporters.

Fatty Acid Degradation/Oxidation

Fatty acids contain a long hydrocarbon chain and a terminal carboxylate group. They are stored in the body as triacyglycerols. The initial event in the utilization of fat as an energy source is the hydrolysis of triacyglycerol emulsion droplets (bile salts emulsify these triacyclycerols) by lipases into fatty acids. Lipase and co-lipase hydrolyze fatty acids in the intestine.  lipoprotein lipase cleaves triglycerides in chylomicrons (transportors of lipids in the blood). lipoprotein lipase requires Apo C-II as an activator. Hormone sensitive lipase sometimes called "triacylglycerol lipase" cleaves triglycerides in adipose tissue. Epinephrine and glucogon induce this lipase by increasing cyclic cAMP which stimulates protein kinase which activates the lipase by phosphorylating it.

Fatty acids are oxidzed in the mitochondrial matrix. However, before they can be oxidized. They are activated on the outer mitochondrial membrane and linked to coenzyme A in a reaction catalyzed by acyl CoA synthetase. Since long chain acyl CoA molecules do not readily traverse the inner mitochondrial membrane, a special transporter carnitine is conjugated to them.

Carnitine palmitoyle transferase I (CPTI) catalyzes the rate limiting step in fatty acid oxidation. CPTI is regulated by malonyl CoA. (malonyl CoA is in turn regulated by the rate limiting step in the synthesis of fatty acids below in that if synthesis of fatty acids is high, [malonyl CoA] is also high which has a negative effect on CPTI. Conversely, in a fasted stated, [malonyl CoA] is low and the negative effect is removed).

The entry of acetyl CoA formed in fatty acid oxidation depends on the availability of oxaloacetate. If there are insufficient carboyhdrates, oxaloacetate will not be made. In such a case, acetyl CoA is diverted from the citric acid cycle to form what are known as "ketone bodies". Ketone bodies are abnormatlly high in untreated diabetics or in people who fast because oxaloacetate is consumed to form glucose in the gluconeogenic pathway and hence is unavailable. The 3 ketone bodies formed are 1) acetoacetate, 2) acetone and 3) B-hydroxybutyrate. Ketone bodies are produced in the liver but used in extrahepatic tissues. The interrelationship between oxydation of fatty acids, the formation of ketone bodies, the citric acid cycle and gluconogenesis is shown below.

Fatty Acid Synthesis

Whereas fatty acid degradation occurs in the mitochondrial matrix, synthesis of fatty acids occurs in the cytosol. Because fatty acid synthesis occurs in the cytosol whereas acetyl Cois formed from pyruvate in mitchondria, acetyl CoA must be transferred from the mitochondria to the cytosol. Since mitochondira are not readily permeable to acetyl CoA, acetyl CoA is condensed with oxaloacetate to form citrate which can pass to the cytosol. Thus citrate is used as a transporter. The citrate is then cleaved by ATP citrate lysase to reform the oxaloacetate and acetyl CoA.

Fatty acid synthesis starts off where fatty acid left off, namely, acetyl CoA. The rate limiting reaction is the carboxylation of acetyl CoA to malonyl CoA in a reaction catalyzed by acetyl CoA carboxylase which has a biotin prosthetic group. Phosphyrlation of acetyl CoA carboxylase results in inactivity whereas dephosphrylation results in activity so that as one might expect, glucogen has a negative regulatory affect and insulin a positive effect upon the activity of this enzyme.

The reductant used in fatty acid synthesis is NADPH whereas it is NAD+ and FAD in fatty acid breakdown. The major product of fatty acid synthesis is palmitate. In eukaryotes, longer fatty acids are actually formed by elongation reaction catalyzed by enzymes on the cytosolic face of the ER membrane through the use of something called the desaturase reaction.

Eicosanoid hormones are derived from polyunsaturated fatty acids. A precursor for eicosanoids is arachidonate which is a fatty acid derived from linoleate (an essential fatty acid) and is the major precursor of several classes of signal molecules - thromboxanes and prostaglandins.

. Amino Acid Catabolism/Degradation

10 of the 20 amino acids required for protein synthesis are essential and must be obtained through ingestion and digestion of dietary protein. Humans required a daily supply of these essential amino acids since there is no storage protein per se as with lipids (triglycerides) and carbohydrates (glycogen). However, in the fasting state, body proteins can be called on for glucose production.

One of the key early steps in amino acid catabolism are transamination reactions which involve the transferring amino acid  nitrogen between amino acids in the body. One of the donor/acceptor pairs in transamination reaction is always glutamate and α-Ketoglutarate. The prostetic group of all aminotransferases is PLP (pyridoxal phosphate) which is derived from vitamin B6.

Transamination reactions provide a mechanism for transfer of amino groups from various amino acids to glutamate which are then oxidatively deaminated via a key enzyme, glutamate dehydrogenase. The products of this reaction are α-Ketoglutarate and ammonia (NH₄).

Ammonia is very toxic. The enzyme, glutamine synthetase catalyzes the conversion of NH₄ to glutamine which is the major transport form of ammonia. In the liver, the reverse reaction is catalyzed by glutaminase, releasing free NH₄⁺ which is then converted to urea, which is the major excreted form of excess nitrogen.

Amino Acid Synthesis

Non-essential amino acids can be synthesized from glycolytic or TCA cycle intermediates or from essential amino acids.

Amino acids are precursors to many important biomolecules. For example, tyrosine is a precursor for catecholamines which are involved as hormones and neurotransmitors (such as epinephrine, norepinephrine, dopamine). Tryptophan is a precursor for serotonin. Catecholamines and serotein are sometimes called "biogenic amines."

Arginine is a precursor for nitric oxide (NO) that is an important regulatory molecule. Arginine is also a precursor for plyamines which carry many + charges and complex with the - P on DNA during DNA replication.

Glutamate is a precursor for GABA and histidine is a precursor for histamine

Nucleotide Biosynthesis

When we think about nucleotides, we think of their necessity for the formation of nucleic acids (DNA & RNA). But nucleotides are also essential for energy metabolism (ATP), signaling and regulatory molecules. In addition, nucleotides are components of coenzymes and serve as activated intermediates. For example, S-adenosylmethionine (SAM) is an activated methyl group donor.

Nucleotide biosynthesis is typically divided into 1) purine biosynthesis and 2) pyrimidine biosynthesis. In both cases, amino acids are necessary.

Purine biosynthesis requires a purine ribose phosphate (PRPP) and glutamine. The reaction is catalyzed by PRPP-Amidotransferase. The reaction is highly regulated with PRPP itself being a + effector.

Pyrimidine biosynthesis also requires amino acids (glutamine as well as asparate). But first the purine ring is formed and only then is the ribose moiety added.

Because nucleotide biosynthesis is an energy consuming process, the body has developed pathways to reuse both purine and pyrimidine bases.

The enzymes ribonucleotide reductase and thymidylate synthase are important enzymes required for the formation of deoxyribunucletide precursors for DNA synthesis. Ribonucleotide reductase converts ribonucletoside diphosphates to deoxyribonucleoside diphosphates. Thymidylate synthesis converts dUMP to dTMP.

Nucleotide Degradation

Purine nucleotide degradation: The final step in purine degration is catalyzed by an enzyme called xanthine oxidase. The final product in this reaction is uric acid which has a limited solubility and can form crystals in tissues if the concentration is too high. This can result in a condition referred to as "gout". Gout can be treated with allopurinol which inhibits uric acid production.

Since deoxyadenosine can only be degraded via the pathway involv. ing the enzyme adenosine deaminase, a deficiency involving adenosine deaminase results in the buildup of deoxyadenosine which can lead to immunodeficiencies.

Pyrimidine nucleotide degradation: Uracil and thymine are degraded via the same pathway of reactions, but the products are different. In the case of uracil, the product is β-alanine and with thymine the product is β-aminoisobutyrate. It is possible to estimate the turnover of DNA by measurement of β-aminoisobutyrate . Levels are increased in patients undergoing chemotherapy or radiation therapy.