Enzyme Kinetics

Enzymology

• Catalytic activity is determined by studying kinetics.
• Kinetics refers to the rate at which an enzyme catalyzes a reaction under experimental conditions.
• Leonor Michaelis and Maud Menten described the mathematical relationship between substrate concentration and enzyme reaction velocity, which is measured by the product formed or substrate consumed over time.
• Turnover number is the simplest measure of an enzyme's catalytic activity.

Terms in Enzyme Kinetics

• Initial velocity (Vo) is the rate at the moment when no product is formed.
• As substrate concentration increases, the enzyme approaches saturation, and the initial velocity reaches a theoretical saturation point called maximal velocity (Vmax).
• Michaelis constant (Km) is the substrate concentration at which the reaction velocity is half of Vmax.
• Km is constant for a specific enzyme, irrespective of substrate or enzyme concentration, and it provides a measure of the enzyme's affinity for the substrate.
• Kinetics is the study of reaction rates and how they change in response to changes in experimental parameters.
• The amount of substrate present is a key factor affecting the rate of enzyme-catalyzed reactions in vitro.

Michaelis-Menten Equation

• This equation defines the quantitative relationship between enzyme reaction rate and substrate concentration: Vo = (Vmax [S]) / (Km + [S]).
  • Vo represents the observed velocity at a given substrate concentration [S].
  • Km is the Michaelis-Menten constant.
  • Vmax is the maximum velocity at saturating substrate concentration [S].
  • Km = (K-1 + K2) / K1.

Lineweaver-Burk Plot

• This plot is a double reciprocal plot and provides a linear representation of enzyme kinetics, making it more accurate and convenient for determining Vmax and Km.
• The equation is derived by taking the reciprocal of both sides of the Michaelis-Menten equation.
• The plot graphs 1/[S] vs. 1/Vo. Slope = Km/Vmax, 1/v = Km/Vmax * 1/[S] + 1/Vmax

Enzyme Inhibitors

• Enzyme inhibitors diminish the velocity of an enzyme-catalyzed reaction.
• These inhibitors include drugs, antibiotics, poisons, and anti-metabolites.
• Enzyme inhibitors help understand enzyme reactions, metabolic regulation, and mechanisms of cell toxicity.
• Enzyme inhibitors form the basis of drug design.
• Enzyme inhibitors act as pharmacologic agents and research tools to study enzyme action mechanisms.
• Inhibitors are classified by site of action, chemical modification of the enzyme, or influence on kinetic parameters.
• Enzyme inhibitors can be reversible or irreversible.

Irreversible Inhibition

• An irreversible inhibitor binds covalently to the enzyme, which results in irrevocable loss of catalytic activity.
• Usually toxic to cells.
• Ions of heavy metals, alkylating agents, and nerve gas poisons are irreversible inhibitors.
• Some insecticides and nerve gases inhibit toxic acetylcholinesterase.
• Irreversible inhibitors can destroy a functional group on an enzyme, that is essential, or they can form stable noncovalent associations.
• Covalent links between an enzyme and an irreversible inhibitor is common.
• For Example : Chymotrypsin reacts with diisopropylfluorophosphate (DIFP).
  • This inhibits the enzyme irreversibly by binding with Ser195 in the active site.
• Acetylcholinesterase is vital for transmitting nerve impulses.
• Inhibition of acetylcholinesterase leads to rapid paralysis and death.
• Chymotrypsin is synthesized as a single polypeptide (chymotrypsinogen).
• It can be activated into three polypeptide chains connected by disulfide bridges.
• The active center consists of a catalytic triad: Ser195, His 57, and Asp 102.

Reversible Inhibitors

• Competitive inhibitor
• Non-competitive inhibitor
• Un-competitive inhibitor

Competitive Inhibition

• Binds directly to the active site and competes with the substrate.
• Increasing substrate levels increases the likelihood of the enzyme colliding with the substrate instead of the inhibitor.
• The maximum rate of enzyme activity (Vmax) can be achieved with enough substrate concentration.

Non-Competitive Inhibition

• Binds to an allosteric site and does not directly compete with the substrate.
• Increasing substrate concentrations does not affect the level of inhibition.
• The maximum rate of enzyme activity (Vmax) is reduced.

Un-competitive Inhibition

• Binds only to the enzyme-substrate complex.
• It does not bind to the free enzyme.
• Inhibitor not overcome by increasing the substrate concentration.
• Both the Km and Vmax are reduced.

Therapeutic Uses of Enzymes

• Systemically used enzymes, including streptokinase and urokinase, increase plasmin levels to achieve thrombolysis, which is used in cases of acute myocardial infarction, acute thrombosis of arteries, deep vein thrombosis, and pulmonary embolism.
• L-Asparaginase is used in acute leukemia and malignant lymphomas, where it hydrolyzes L-Asparagine and inhibits tumor cell growth.
• Digestive enzymes Amylase, lipase and protease can provide replacement therapy in pancreatic insufficiency.
• a-chymotrypsin is used as an adjunct therapy in the management of inflammatory edema due to injury, postsurgical infections, and dental procedures.
• Serratopeptidase provides fibrinolytic activity and reduces inflammation in traumatic injury and after operations, and it can treat subconjunctival bleeding.
• Locally used enzymes, such as hyaluronidase, breaks down ground matter which promotes diffusion of fluids given subcutaneously and is used as an intra-articular injection for osteoarthritis pain relief.

Diagnostic Significance of Enzymes

• Clinically important enzymes
• Increased levels of:
  • Lactate dehydrogenase (LDH) indicates myocardial infarction, myopathy, or muscle disorder; also seen in leukemias, acute hepatitis, and carcinomatosis.
  • Aspartyl transaminase (AST) or Serum glutamyl oxaloacetate transaminase (SGOT) indicates myocardial infarction.
  • Alanine transaminase (ALT) or Serum glutamyl pyruvate transaminase (SGPT) indicates liver disorders.
  • Creatine phosphokinase (CPK) indicates myocardial infarction and myopathy.
  • Alkaline phosphatase indicates bone disorders, obstructive jaundice, and hyperparathyroidism.
  • Acid phosphatase indicates prostrate carcinoma.
  • Isocitrate dehydrogenase indicates Brain tumor and meningitis, liver diseases.
  • Amylase indicates Pancreatitis, parotitis, intestinal obstruction, diabetes
  • Lipase indicates Pancreatitis or carcinoma of pancreas.
  • Gamma glutamyl transpeptidase (g-GT) indicates liver damage.

Lipids

• Lipids are relatively insoluble in water but soluble in nonpolar organic solvents like benzene, chloroform, ether, hot alcohol, and acetone.
• They are hydrophobic or amphiphilic molecules.
• Organic compounds mostly formed of alcohol and fatty acids joined by ester linkages.
• Differ from carbohydrates, proteins, and nucleic acids by not being huge molecules and not being built of monomers.
• They exist as waxy, greasy, or oily compounds in plants and animals.
• Lipids share the common property of being hydrophobic.
• They contain hydrophobic regions and consist a small number of molecules in their backbone
• Fatty acids are carboxylic acids with hydrocarbon chains ranging from 4 to 36 carbons.
• Some fatty acids have unbranched and fully saturated chains. others contain one or more double bonds.

Structure and function of Lipids

• Includes representative lipids formed through the combination of glycerol and fatty acids.
• Triglycerides are the most abundant family of lipids in plant and animal cells.