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.

Acids and bases can be classified as proton donors and proton acceptors, respectively. This means that the conjugate base of a given acid will carry a net charge that is more negative than the corresponding acid. In biologically relavent compounds various weak acids and bases are encountered, e.g. the acidic and basic amino acids, nucleotides, phospholipids etc.

Weak acids and bases in solution do not fully dissociate and, therefore, there is an equilibrium between the acid and its conjugate base. This equilibrium can be calculated and is termed the equilibrium constant = K_a. This is also  referred to as the dissociation constant as it pertains to the dissociation of protons from acids and bases.

In the reaction of a weak acid:

HA <-----> A^- + H⁺

the equlibrium constant can be calculated from the following equation:

K_a = [H⁺][A^-]/[HA]

As in the case of the ion product:

pK_a = -logK_a

Therefore, in obtaining the -log of both sides of the equation describing the dissociation of a weak acid we arrive at the following equation:

-logK_a = -log[H⁺][A^-]/[HA]

Since as indicated above -logK_a = pK_a and taking into account the laws of logrithms:

pK_a = -log[H⁺] -log[A^-]/[HA]

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

From this equation it can be seen that the smaller the pK_a value the stronger is the acid. This is due to the fact that the stronger an acid the more readily it will give up H⁺ and, therefore, the value of [HA] in the above equation will be relatively small.

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

VITAMIN C: ASCORBIC ACID, ASCORBATE

Vitamin C benefits the body by holding cells together through collagen synthesis; collagen is a connective tissue that holds muscles, bones, and other tissues together. Vitamin C also aids in wound healing, bone and tooth formation, strengthening blood vessel walls, improving immune system function, increasing absorption and utilization of iron, and acting as an antioxidant.

RDA The Recommended Dietary Allowance (RDA) for Vitamin C is 90 mg/day for adult males and 75 mg/day for adult females

Vitamin C Deficiency

Severe vitamin C deficiency result in the disease known as scurvy, causing a loss of collagen strength throughout the body. Loss of collagen results in loose teeth, bleeding and swollen gums, and improper wound healing.

Erythrocytes and the Pentose Phosphate Pathway

The predominant pathways of carbohydrate metabolism in the red blood cell (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate (2,3-BPG) metabolism (refer to discussion of hemoglobin for review of the synthesis and role role of 2,3-BPG).

Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of methemoglobin. The PPP supplies the RBC with NADPH to maintain the reduced state of glutathione.

The inability to maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H₂O₂, that in turn results in a weakening of the cell wall and concomitant hemolysis.

Accumulation of H₂O₂ also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell wall.

Glutathione removes peroxides via the action of glutathione peroxidase.

The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH.

Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.