Enzyme Kinetics

Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process. They achieve their effect by temporarily binding to the substrate and, in doing so, lowering the activation energy needed to convert it to a product.

The rate at which an enzyme works is influenced by several factors, e.g.,

• the concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in unit of molarity.
• the temperature. As the temperature rises, molecular motion - and hence collisions between enzyme and substrate - speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective.
• the presence of inhibitors.
  • competitive inhibitors are molecules that bind to the same site as the substrate - preventing the substrate from binding as they do so - but are not changed by the enzyme.
  • noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power.
• pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.

The study of the rate at which an enzyme works is called enzyme kinetics.

Factors regulating blood calcium level

(i) Vitamin D

(a) Vitamin D and absorption of calcium: Active form of calcium is calcitriol. Calcitriol enters intestinal wall and binds to cytoplasmic receptor and then binds with DNA causes depression and consequent transcription of gene code for calbindin. Due to increased availability of calbindin, absorption of calcium increases leading to increased blood calcium level.
(b) Vitamin D and Bone: Vitamin D activates osteoblast, bone forming cells & also stimulates secretion of alkaline phosphatase. Due to this enzyme, calcium and phosphorus increase.

(c) Vitamin D and Kidney: Calcitriol increase reabsorption of calcium and phosphorus by renal tubules.

(ii) Parathyroid  hormone (PTH)

Normal PTH level in serum is 10-60ng/l.

(a) PTH and bones: In bone, PTH causes demineralization. It also causes recreation of collagenase from osteoclast  leads to loss of matrix and bone resorption. As a result, mucopolysacharides and hydroxyproline are excreted in urine.

(b) PTH and Kidney: In kidney, PTH causes increased reabsorption of calcium but decreases reabsorption of phosphorus from kidney tubules.

(iii) Calcitonin Calcitonin decreases serum calcium level. It inhibits resorption of bone. It decreases the activity of osteoclasts and increases osteoblasts.

Hyper Calcemia When plasma Ca2+ level is more than 11mg/dl is called Hypercalcemia. It is due to parathyroid adenoma or ectopic PTH secreting tumor. calcium excreted in urine decreases excretion of chloride causing hyperchloremic acidosis.

Hypocalcemia Plasma calcium level less than 8mg/dl is called hypocalcemia. Tetany due to accidental surgical removal of parathyroid glands or by autoimmune disease. In tetany, neuromuscular irritability is increased. Increased Q-7 internal in ECG is seen. Main manifestation is carpopedal spasm. Laryngismus and stridor are also observed.

FACTORS AFFECTING ENZYME ACTIVITY

Velocity or rate of enzymatic reaction is assessed by the rate of change in concentration of substrate or product at a given time duration. Various factors which affect the activity of enzymes include:

1. Substrate concentration

2. Enzyme concentration

3. Product concentration

4. Temperature 5. Hydrogen ion concentration (pH)

6. Presence of activators

7. Presence of inhibitor

Effect of substrate Concentration :  Reaction velocity of an enzymatic process increases with constant enzyme concentration and increase in substrate concentration.

Effect of enzyme Concentration: As there is optimal substrate concentration, rate of an enzymatic reaction or velocity (V) is directly proportional to the enzyme concentration.

Effect of product concentration In case of a reversible reaction catalyzed by a enzyme, as per the law of mass action the rate of reaction is slowed down with equilibrium. So, rate of reaction is slowed, stopped or even reversed with increase in product concentration

Effect of temperature: Velocity of enzymatic reaction increases with temperature of the medium which they are most efficient and the same is termed as optimum temperature.

Effect of pH: Many enzymes are most efficient in the region of pH 6-7, which is the pH of the cell. Outside this range, enzyme activity drops off very rapidly. Reduction in efficiency caused by changes in the pH is due to changes in the degree of ionization of the substrate and enzyme.

Highly acidic or alkaline conditions bring about a denaturation and subsequent loss of enzymatic activity

Exceptions such as pepsin (with optimum pH 1-2), alkaline phosphatase (with optimum pH 9-10) and acid phosphatase (with optimum pH 4-5)

Presence of activators Presence of certain inorganic ions increases the activity of enzymes. The best examples are chloride ions activated salivary amylase and calcium activated lipases.

Effect of Inhibitors The catalytic enzymatic reaction may be inhibited by substances which prevent the formation of a normal enzyme-substrate complex. The level of inhibition then depends entirely upon the relative concentrations of the true substrate and the inhibitor

Glycogen Metabolism

The formation of glycogen from glucose is called Glycogenesis

Glycogen is a polymer of glucose residues linked mainly by a(1→ 4)  glycosidic linkages. There are a(1→6) linkages at branch points. The chains and branches are longer than shown. Glucose is stored as glycogen predominantly in liver and muscle cells

Glycogen Synthesis

Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis. As glucose residues are added to glycogen, UDP-glucose is the substrate and UDP is released as a reaction product. Nucleotide diphosphate sugars are precursors also for synthesis of other complex carbohydrates, including oligosaccharide chains of glycoproteins, etc.

UDP-glucose is formed from glucose-1-phosphate and uridine triphosphate (UTP)

glucose-1-phosphate + UTP → UDP-glucose + 2 P_i

Cleavage of PPi is the only energy cost for glycogen synthesis (1P bond per glucose residue)

Glycogenin initiates glycogen synthesis. Glycogenin is an enzyme that catalyzes glycosylation of one of its own tyrosine residues.

Physiological regulation of glycogen metabolism

Both synthesis and breakdown of glycogen are spontaneous. If glycogen synthesis and phosphorolysis were active simultaneously in a cell, there would be a futile cycle with cleavage of 1 P bond per cycle

To prevent such a futile cycle, Glycogen Synthase and Glycogen Phosphorylase are reciprocally regulated, both by allosteric effectors and by covalent modification (phosphorylation)

Glycogen catabolism (breakdown)

Glycogen Phosphorylase catalyzes phosphorolytic cleavage of the →(1→4) glycosidic linkages of glycogen, releasing glucose-1-phosphate as the reaction product.

Glycogen (n residues) + P_i → glycogen (n-1 residues) + glucose-1-phosphate

The Major product of glycogen breakdown is glucose -1-phosphate

Fate of glucose-1-phosphate in relation to other pathways:

Phosphoglucomutase catalyzes the reversible reaction:

Glucose-1-phosphate → Glucose-6-phosphate

Glucagon

Glucagon, a peptide hormone synthesized and secreted from the α-cells of the islets of Langerhans of pancreas, raises blood glucose levels. The pancreas releases glucagon when blood sugar (glucose) levels fall too low. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. Glucagon and insulin are part of a feedback system that keeps blood glucose levels at a stable level.

Regulation and function

Secretion of glucagon is stimulated by hypoglycemia, epinephrine, arginine, alanine, acetylcholine, and cholecystokinin.

Secretion of glucagon is inhibited by somatostatin, insulin, increased free fatty acids and keto acids into the blood, and increased urea production.

By rearranging the above equation we arrive at the Henderson-Hasselbalch equation:

pH = pK_a + log[A^-]/[HA]

It should be obvious now that the pH of a solution of any acid (for which the equilibrium constant is known, and there are numerous tables with this information) can be calculated knowing the concentration of the acid, HA, and its conjugate base [A^-].

At the point of the dissociation where the concentration of the conjugate base [A^-] = to that of the acid [HA]:

pH = pK_a + log[1]

The log of 1 = 0. Thus, at the mid-point of a titration of a weak acid:

pK_a = pH

In other words, the term pK_a is that pH at which an equivalent distribution of acid and conjugate base (or base and conjugate acid) exists in solution.