The Effects of Enzyme Inhibitors

Enzymes can be inhibited

• competitively, when the substrate and inhibitor compete for binding to the same active site or
• noncompetitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency.

The distinction can be determined by plotting enzyme activity with and without the inhibitor present.

Competitive Inhibition

In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while V_max can still be reached if sufficient substrate is available, one-half V_max requires a higher [S] than before and thus K_m is larger.

Noncompetitive Inhibition

With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out

• enzyme rate (velocity) is reduced for all values of [S], including
• V_max and one-half V_max but
• K_m remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.

BIOLOGICAL BUFFER SYSTEMS 

Cells and organisms maintain a specific and constant cytosolic pH, keeping biomolecules in their optimal ionic state, usually near pH 7. In multicelled organisms, the pH of the extracellular fluids (blood, for example) is also tightly regulated. Constancy of pH is achieved primarily by biological buffers : mixtures of weak acids and their conjugate bases 

Body fluids and their principal buffers

Body fluids                     Principal buffers

Extracellular fluids        {Biocarbonate buffer Protein buffer } 

Intracellular fluids         {Phosphate buffer, Protein }

Erythrocytes                 {Hemoglobin buffer}

Insulin

Insulin is a polypeptide hormone synthesized in the pancreas by β-cells, which construct a single chain molecule called proinsulin. 

Insulin, secreted by the β-cells of the pancreas in response to rising blood glucose levels, is a signal that glucose is abundant.

Insulin binds to a specific receptor on the cell surface and exerts its metabolic effect by a signaling pathway that involves a receptor tyrosine kinase phosphorylation cascade.

The pancreas secretes insulin or glucagon in response to changes in blood glucose.

Each cell type of the islets produces a single hormone: α-cells produce glucagon; β-cells, insulin; and δ-cells, somatostatin.

Insulin secretion

When blood glucose rises, GLUT2 transporters carry glucose into the b-cells, where it is immediately converted to glucose 6-phosphate by hexokinase IV (glucokinase) and enters glycolysis. The increased rate of glucose catabolism raises [ATP], causing the closing of ATP-gated K+ channels in the plasma membrane. Reduced efflux of K+ depolarizes the membrane, thereby opening voltage-sensitive Ca2+ channels in the plasma membrane. The resulting influx of Ca2+ triggers the release of insulin by exocytosis.

Insulin lowers blood glucose by stimulating glucose uptake by the tissues; the reduced blood glucose is detected by the β-cell as a diminished flux through the hexokinase reaction; this slows or stops the release of insulin. This feedback regulation holds blood glucose concentration nearly constant despite large fluctuations in dietary intake.

Insulin counters high blood glucose

Insulin stimulates glucose uptake by muscle and adipose tissue, where the glucose is converted to glucose 6-phosphate. In the liver, insulin also activates glycogen synthase and inactivates glycogen phosphorylase, so that much of the glucose 6-phosphate is channelled into glycogen.

Diabetes mellitus, caused by a deficiency in the secretion or action of insulin, is a relatively common disease. There are two major clinical classes of diabetes mellitus: type I diabetes, or insulin-dependent diabetes mellitus (IDDM), and type II diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), also called insulin-resistant diabetes. In type I diabetes, the disease begins early in life and quickly becomes severe. IDDM requires insulin therapy and careful, lifelong control of the balance between dietary intake and insulin dose.

Characteristic symptoms of type I (and type II) diabetes are excessive thirst and frequent urination (polyuria), leading to the intake of large volumes of water (polydipsia)

Type II diabetes is slow to develop (typically in older, obese individuals), and the symptoms are milder.

Amino Acid Biosynthesis

Glutamate and Aspartate

Glutamate and aspartate are synthesized from their widely distributed a-keto acid precursors by simple 1-step transamination reactions. The former catalyzed by glutamate dehydrogenase and the latter by aspartate aminotransferase, AST. Aspartate is also derived from asparagine through the action of asparaginase. The importance of glutamate as a common intracellular amino donor for transamination reactions and of aspartate as a precursor of ornithine for the urea cycle is described in the Nitrogen Metabolism page.
 

Alanine and the Glucose-Alanine Cycle

Role in protein synthesis,

Alanine is second only to glutamine in prominence as a circulating amino acid.. When alanine transfer from muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is recycled and most nitrogen eliminated.

There are 2 main pathways to production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (also referred to as serum glutamate-pyruvate transaminase, SGPT).

glutamate + pyruvate <-------> a-KG + alanine

Cysteine Biosynthesis

The sulfur for cysteine synthesis comes from the essential amino acid methionine. A condensation of ATP and methionine catalyzed by methionine adenosyltransferase yields S-adenosylmethionine

Tyrosine Biosynthesis

Tyrosine is produced in cells by hydroxylating the essential amino acid phenylalanine. This relationship is much like that between cysteine and methionine. Half of the phenylalanine required goes into the production of tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine are reduced by about 50%.

Phenylalanine hydroxylase is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine. The reductant is the tetrahydrofolate-related cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADH-dependent enzyme dihydropteridine reductase (DHPR).

Ornithine and Proline Biosynthesis

Glutamate is the precursor of both proline and ornithine, with glutamate semialdehyde being a branch point intermediate leading to one or the other of these 2 products. While ornithine is not one of the 20 amino acids used in protein synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the urea cycle

Serine Biosynthesis

The main pathway to serine starts with the glycolytic intermediate 3-phosphoglycerate. An NADH-linked dehydrogenase converts 3-phosphoglycerate into a keto acid, 3-phosphopyruvate, suitable for subsequent transamination. Aminotransferase activity with glutamate as a donor produces 3-phosphoserine, which is converted to serine by phosphoserine phosphatase.
 

Glycine Biosynthesis

The main pathway to glycine is a 1-step reaction catalyzed by serine hydroxymethyltransferase. This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N⁵,N¹⁰-methylene-THF. Glycine produced from serine or from the diet can also be oxidized by glycine cleavage complex, GCC, to yield a second equivalent of N⁵,N¹⁰-methylene-tetrahydrofolate as well as ammonia and CO₂.

Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine.

Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis

Glutamate is synthesized by the reductive amination of a-ketoglutarate catalyzed by glutamate dehydrogenase; it is thus a nitrogen-fixing reaction. In addition, glutamate arises by aminotransferase reactions, with the amino nitrogen being donated by a number of different amino acids. Thus, glutamate is a general collector of amino nitrogen.

Aspartate is formed in a transamintion reaction catalyzed by aspartate transaminase, AST. This reaction uses the aspartate a-keto acid analog, oxaloacetate, and glutamate as the amino donor. Aspartate can also be formed by deamination of asparagine catalyzed by asparaginase.

Asparagine synthetase and glutamine synthetase, catalyze the production of asparagine and glutamine from their respective a-amino acids. Glutamine is produced from glutamate by the direct incorporation of ammonia; and this can be considered another nitrogen fixing reaction. Asparagine, however, is formed by an amidotransferase reaction.

Aminotransferase reactions are readily reversible. The direction of any individual transamination depends principally on the concentration ratio of reactants and products. By contrast, transamidation reactions, which are dependent on ATP, are considered irreversible. As a consequence, the degradation of asparagine and glutamine take place by a hydrolytic pathway rather than by a reversal of the pathway by which they were formed. As indicated above, asparagine can be degraded to aspartate

Folate: Folic Acid, Folacin Folate, also known as folic acid or folacin, aids in protein metabolism, promoting red blood cell formation, and lowering the risk for neural tube birth defects. Folate may also play a role in controlling homocysteine levels, thus reducing the risk for coronary heart disease.

RDA for folate is 400 mcg/day for adult males and females. Pregnancy will increase the RDA for folate to 600 mcg/day.

Folate Deficiency

Folate deficiency affects cell growth and protein production, which can lead to overall impaired growth. Deficiency symptoms also include anemia and diarrhea.

A folate deficiency in women who are pregnant or of child bearing age may result in the delivery of a baby with neural tube defects such as spina bifida.

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.