Enzymes are protein catalyst produced by a cell and responsible ‘for the high rate’ and specificity of one or more intracellular or extracellular biochemical reactions.

Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Enzyme reactions are always reversible.

The substance, upon which an enzyme acts, is called as substrate. Enzymes are involved in conversion of substrate into product.

Almost all enzymes are globular proteins consisting either of a single polypeptide or of two or more polypeptides held together (in quaternary structure) by non-covalent bonds. Enzymes do nothing but speed up the rates at which the equilibrium positions of reversible reactions are attained.

 In terms of thermodynamics, enzymes reduce the activation energies of reactions, enabling them to occur much more readily at low temperatures - essential for biological systems.

CLINICAL SIGNIFICANCE OF ENZYMES

The measurement of enzymes level in serum is applied in diagnostic application

Pancreatic Enzymes

Acute pancreatitis is an inflammatory process where auto digestion of gland was noticed with activation of the certain pancreatic enzymes. Enzymes which involves in pancreatic destruction includes α-amylase, lipase etc.,

1.  α-amylase (AMYs) are calcium dependent hydrolyase class  of metaloenzyme that catalyzes the hydrolysis of 1, 4- α-glycosidic linkages in polysaccharides. The normal values of amylase is in range of 28-100 U/L. Marked increase of 5 to 10 times the upper reference limit (URL) in AMYs activity indicates acute pancreatitis and severe glomerular impairment.

2.  Lipase is single chain glycoprotein. Bile salts and a cofactor called colipase are required for full catalytic activity of lipase. Colipase is secreted by pancreas. Increase in plasma lipase activity indicates acute pancreatitis and carcinoma of the pancreas.

Liver Enzymes

Markers of Hepatocellular Damage

1.  Aspartate transaminase (AST) Aspartate transaminase is present in high concentrations in cells of cardiac and skeletal muscle, liver, kidney and erythrocytes. Damage to any of these tissues may increase plasma AST levels.

The normal value of AST for male is <35 U/ L and for female it is <31 U/L.

2.  Alanine transaminase (ALT) Alanine transaminase is present at high concentrations in liver and to a lesser extent, in skeletal muscle, kidney and heart. Thus in case of liver damage increase in both AST and ALT were noticed. While in myocardial infarction AST is increased with little or no increase in ALT.

The normal value of ALT is <45 U/L and <34 U/L for male and female respectively

Markers of cholestasis

1.  Alkaline phosphatases

Alkaline phosphatases are a group of enzymes that hydrolyse organic phosphates at high pH. They are present in osteoblasts of bone, the cells of the hepatobiliary tract, intestinal wall, renal tubules and placenta.

Gamma-glutamyl-transferase (GGT) Gamma-glutamyl-transferase catalyzes the transfere of the γ–glutamyl group from peptides. The activity of GGT is higher in men than in women. In male the normal value of GGT activity is <55 U/L and for female it is <38 U/L.

2.  Glutamate dehydrogenase (GLD) Glutamate dehydrogenase is a mitochondrial enzyme found in liver, heart muscle and kidneys.

Muscle Enzymes

1.  Creatine Kinase Creatine kinase (CK) is most abundant in cells of brain, cardiac and skeletal.

2.  Lactate Dehydrogenase

Lactate dehydrogenase (LD) catalyses the reversible interconversion of lactate and pyruvate.

IRON

The normal limit for iron consumption is 20 mg/day for adults, 20-30 mg/day for children and 40 mg/day for pregnant women.

Milk is considered as a poor source of iron.

Factors influencing absorption of iron Iron is absorbed by upper part of duodenum and is affected by various factors

(a) Only reduced form of iron (ferrous) is absorbed and ferric form are not absorbed

 (b) Ascorbic acid (Vitamin C) increases the absorption of iron (c) The interfering substances such as phytic acid and oxalic acid decreases absorption of iron

Regulation of absorption of Iron

Absorption of iron is regulated by three main mechanisms, which includes

(a) Mucosal Regulation

(b) Storer regulation

(c) Erythropoietic regulation

In mucosal regulation absorption of iron requires DM-1 and ferroportin. Both the proteins are down regulated by hepcidin secreted by liver. The above regulation occurs when the body irons reserves are adequate. When the body iron content gets felled, storer regulation takes place. In storer regulation the mucosal is signaled for increase in iron absorption. The erythropoietic regulation occurs in response to anemia. Here the erythroid cells will signal the mucosa to increase the iron absorption.

Iron transport in blood

The transport form of iron in blood is transferin. Transferin are glycoprotein secreted by liver. In blood, the ceruloplasmin is the ferroxidase which oxidizes ferrous to ferric state.

Storage form of iron is ferritin. Almost no iron is excreted through urine.

Anemia

Anemia is the most common nutritional deficiency disease. The microscopic appearance of anemia is characterized by microcytic hypochromic anemia

The abnormal gene responsible for hemosiderosis is located on the short arm of chromosome No.6.

The main causes of iron deficiency or anemia are

(a) Nutritional deficiency of iron (b) Lack of iron absorption (c) Hook worm infection (d) Repeated pregnancy (e) Chronic blood loss (f) Nephrosis (g) Lead poisoning

Acyl-CoA Synthases (Thiokinases), associated with endoplasmic reticulum membranes and the outer mitochondrial membrane, catalyze activation of long chain fatty acids, esterifying them to coenzyme A, as shown at right. This process is ATP-dependent, and occurs in 2 steps. There are different Acyl-CoA Synthases for fatty acids of different chain lengths. 

Exergonic hydrolysis of PP_i (P~P), catalyzed by Pyrophosphatase, makes the coupled reaction spontaneous. Overall, two ~P bonds of ATP are cleaved during fatty acid activation. The acyl-coenzyme A product includes one "high energy" thioester linkage.

Summary of fatty acid activation:

• fatty acid + ATP → acyl-adenylate + PP_i
  PP_i  → P_i
• acyladenylate + HS-CoA → acyl-CoA + AMP

Overall: fatty acid + ATP + HS-CoA → acyl-CoA + AMP +  2 P_i

For most steps of the b-Oxidation Pathway, there are multiple enzymes specific for particular fatty acid chain lengths.

Fatty acid b-oxidation is considered to occur in the mitochondrial matrix. Fatty acids must enter the matrix to be oxidized. However enzymes of the pathway specific for very long chain fatty acids are associated with the inner mitochondrial membrane (facing the matrix).

Fatty acyl-CoA formed outside the mitochondria can pass through the outer mitochondrial membrane, which contains large VDAC channels, but cannot penetrate the mitochondrial inner membrane.

Transfer of the fatty acid moiety across the inner mitochondrial membrane involves carnitine.

Carnitine Palmitoyl Transferases catalyze transfer of a fatty acid between the thiol of Coenzyme A and the hydroxyl on carnitine.

Carnitine-mediated transfer of the fatty acyl moiety into the mitochondrial matrix is a 3-step process, as presented below.

1. Carnitine Palmitoyl Transferase I, an enzyme associated with the cytosolic surface of the outer mitochondrial membrane, catalyzes transfer of a fatty acid from ester linkage with the thiol of coenzyme A to the hydroxyl on carnitine.
2. Carnitine Acyltransferase, an antiporter in the inner mitochondrial membrane, mediates transmembrane exchange of fatty acyl-carnitine for carnitine.
3. Within the mitochondrial matrix (or associated with the matrix surface of the inner mitochondrial membrane, Carnitine Palmitoyl Transferase II catalyzes transfer of the fatty acid from carnitine to coenzyme A. (Carnitine exits the matrix in step 2.) The fatty acid is now esterified to coenzyme A within the mitochondrial matrix

Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria.

Malonyl-CoA inhibits Carnitine Palmitoyl Transferase I. (Malonyl-CoA is also a precursor for fatty acid synthesis). Malonyl-CoA is produced from acetyl-CoA by the enzyme Acetyl-CoA Carboxylase

AMP-Activated Kinase, a sensor of cellular energy levels, catalyzes phosphorylation of Acetyl-CoA Carboxylase under conditions of high AMP (when ATP is low). Phosphorylation inhibits Acetyl-CoA Carboxylase, thereby decreasing malonyl-CoA production.

The decrease in malonyl-CoA concentration releases Carnitine Palmitoyl Transferase I from inhibition. The resulting increase in fatty acid oxidation generates acetyl-CoA for entry into Krebs cycle, with associated production of ATP

Thyroid Hormones

Thyroid hormones (T4 and T3) are tyrosine-based hormones produced by the follicular cells of the thyroid gland and are regulated by TSH made by the thyrotropes of the anterior pituitary gland, are primarily responsible for regulation of metabolism. Iodine is necessary for the production of T3 (triiodothyronine) and T4 (thyroxine).

A deficiency of iodine leads to decreased production of T3 and T4, enlarges  the thyroid tissue and will cause the disease known as goitre.

Thyroid hormones are transported by Thyroid-Binding Globulin

Thyroxine binding globulin (TBG), a glycoprotein binds T4 and T3 and has the capacity to bind 20 μg/dL of plasma.

Diseases

1. Hyperthyroidism (an example is Graves Disease) is the clinical syndrome caused by an excess of circulating free thyroxine, free triiodothyronine, or both. It is a common disorder that affects approximately 2% of women and 0.2% of men.

2 Hypothyroidism (an example is Hashimoto’s thyroiditis) is the case where there is a deficiency of thyroxine, triiodiothyronine, or both.

CLASSIFICATION OF LIPIDS

Lipids are classified as follows:

1. Simple lipids: Esters of fatty acids with various alcohols.

(a) Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state. A long-chain carboxylic acid; those in animal fats and vegetable oils often have 12–22 carbon atoms.

(b) Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols. Waxes are carboxylic acid esters, RCOOR’ ,with long, straight hydrocarbon chains in both R groups

2. Complex lipids: Esters of fatty acids containing groups in addition to an alcohol and a fatty acid.

(a) Phospholipids: Lipids containing, in addition to fatty acids and an alcohol, a phosphoric acid residue. They frequently have nitrogen containing bases and other substituents,

Eg  glycerophospholipids the alcohol is glycerol

     sphingophospholipids the alcohol is sphingosine.

(b) Glycolipids (glycosphingolipids): Lipids containing a fatty acid, sphingosine, and carbohydrate. These lipids contain a fatty acid, carbohydrate and nitrogenous base. The alcohol  is sphingosine, hence they are also called as glycosphingolipids. Clycerol  and phosphate  are absent  

e.g., cerebrosides, gangliosides.

(c) Other complex lipids: Lipids such as sulfolipids and aminolipids. Lipoproteins may also be placed in this category.

3. Precursor and derived lipids: These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid soluble vitamins, and hormones. Because they are uncharged, acylglycerols (glycerides), cholesterol, and cholesteryl esters are termed neutral lipids

4. Miscellaneous lipids: These include a large number of compounds possessing the characteristics of lipids e.g., carotenoids, squalene, hydrocarbons such as pentacosane (in bees wax), terpenes etc.

NEUTRAL LIPIDS: The lipids which are uncharged are referred to as neutral lipids. These are mono-, di-, and triacylglycerols, cholesterol and cholesteryl esters.