Enzymes in Physical Therapy: Properties, Mechanism, and Clinical Significance

Enzymes Physical therapy Level I Prof. Fathia El Sharkawi Definition • Enzymes are biologically active proteins within the cell. They accelerate all the biological reactions that occur in biological systems without appearing in the final product. • Enzyme+ substrate ↔ Enzyme- substrate complex. • [ES] ↔ Enzyme + Product Properties? • Highly specific Unchanged after reaction • Can accelerate reactions >106 fold • Turnover • Cannot alter reaction equilibria What do enzymes do? • Enzymes provide support for many important processes within the body. Some examples include: The digestive system: Enzymes help the body break down larger complex molecules into smaller molecules, such as glucose, so that the body can use them as fuel. What do enzymes do? • DNA replication: Each cell in the body contains DNA. Each time a cell divides, the cell needs to copy its DNA. Enzymes help in this process by unwinding the DNA coils. • Liver enzymes: The liver breaks down toxins in the body. To do this, it uses a range of enzymes the facilitate the process of destroying the toxins. What do enzymes do? • Other activities enzymes help with include: • hormone production • cell regulation • creating movement to make the muscle contract • transporting materials around a cell • respiration Cofactors • Some enzymes cannot function unless they attach to a specific non-protein molecule, known as cofactors. There are two types Source of cofactors, ions and coenzymes. • Ions are inorganic molecules that loosely bond to the enzyme to ensure it can function such as Fe+2, Mg+2, Mn+2, or Zn+2 • Coenzymes are organic molecules that also loosely bond with and allow an enzyme to do its job as vitamins or compound derived from vitamins. • When a cofactor bonds tightly with an enzyme, it is known as a prosthetic group. Activity of enzymes • Active site Enzyme molecules contain a special pocket or cleft called the active sites (region of enzyme where reactions occur). • The active site contains amino acid side chains( R) that create a three dimensional surface complementary to the substrate. • Some enzymes associates with a non protein cofactor needed for enzymatic activity. This includes as metal ions as zn, Fe and organic molecules called coenzymes that are often derivatives of vitamins( NAD, FAD coenzyme A). How are enzymes work? Mechanism of action • 1- Key and Lock Theory: • This theory postulates that the active site of the enzyme has fixed shape ie: the enzyme is preshaped before subs.binding. • But this theory failed to explaine how molecules that are unrelated in structure to subs. Or active site can stimulate or inhibit an enzyme action. How are enzymes work? • 2- The induced Fit model of Koshland: • The active sites of enzymes have shapes that are complementary to that of substrate only after the substrate is bound. • The substrate induces a conformational change in the enzyme. This allows amino acid residues or other groups on the enzyme in the correct orientation for substrate binding Induced conformational change in hexokinase Mathews, Van Holde and Ahern (3rd edition) Enzyme Nomenclature • Enzymes are named according to the type of reaction they catalyze and/or their substrate Substrate = the reactant upon which the specific enzyme acts – Enzyme physically binds to the substrate Enzyme Substrate Enzyme/substrate complex Enzyme Nomenclature • Suffix of an enzyme –ase – Lactase, amylase, lipase or protease denotes an enzyme • Some digestive enzymes have the suffix –in – Pepsin, trypsin & chymotrypsin . • Prefix denotes the type of reaction the enzyme catalyzes: –Oxidase: redox reaction – Hydrolase: Addition of water to break one component into two parts • Substrate identity is often used together with the reaction type – Pyruvate carboxylase, lactate dehydrogenase. Examples of specific enzymes • Lipases: This group of enzymes help digest fats in the gut. • Amylase: In the saliva, amylase helps change starches into sugars. • Maltase: This also occurs in the saliva, and breaks the sugar maltose into glucose. • Trypsin: These enzymes break proteins down into amino acids in the small intestine. • Lactase: Lactase breaks lactose, the sugar in milk, into glucose and galactose. • Acetylcholinesterase: These enzymes break down the neurotransmitter acetylcholine in nerves and muscles. • Helicase: Helicase enzymes unravel ( untie) DNA. • DNA polymerase: These enzymes synthesize DNA from deoxyribonucleotides. Classification of Enzymes • 1-Oxidoreductase: Redox reaction (reduction & oxidation) • 2-Transferase :Transfer of a functional group from 1 molecule to another. • 3-Hydrolase :Hydrolysis reaction • 4-Isomerase: Transfer groups within molecules to form isomer. • 5- Lyase :Addition / removal of atoms to / from double bond • 6-Ligase: catalyze the junction of two molecules. Oxidoreductase Enzymes in this category include : • Dehydrogenases • Reductases • Oxidases • Peroxidases. EXAMPLES OF OXIDOREDUCTASES • Lactate dehydrogenase (LDH) • Glucose 6-phosphate dehydrogenase (G-6-PD) • Cytochrome oxidase • Alcohol Dehydrogenase Transferases Enzymes in this category include : • Amino transferase or Transaminase • Kinase: catalyzes the transfer of phosphate groups EXAMPLES OF TRANSFERASE • Aspartate amino transaminase(AST) • Alanine amino transaminase (ALT) • Hexokinase • Transmethylase Hydrolases ENZYMES IN THIS CATEGORY ARE: • All digestive enzymes like - α-amylase, - Pepsin, - Trypsin, - Chymotrypsin, - Lipase • Acid phosphatase and alkaline phosphatase. Lyases Lyases catalyze the cleavage of Carbon-Oxygen(C-O), CarbonCarbon(C-C) and Carbon-Nitrogen (C-N) bonds by means other than hydrolysis or oxidation, giving rise to compound with double bonds or catalyze the reverse reaction, by the addition of group to a double bond. • Aldolase • Fumarase • Argininosuccinase • Carbonic anhydrase • HMG CoA Lyases Isomerases EXAMPLES OF ISOMERASES - Phosphoglucomutase - Triphosphate isomerase - Phosphohexose isomerase Ligases (Synthetases) Ligases catalyze the joining of two molecules coupled with the hydrolysis of ATP. A + B + ATP ➞ A B+ ADP + Pi EXAMPLES OF LIGASES • Glutamine synthetase • Pyruvate carboxylase • DNA ligases Factors Affecting Enzymatic Activity 1. Enzyme concentration 2. Temperature 3. Hydrogen ion concentration or pH 4. Substrate concentration 5. Inhibitors 6. Product concentration 7. Activators 8. Physical agents Factors Affecting Enzymatic Activity • 1- Enzyme Concentration: • V ά .to E.at the beginning of reaction, by time the rate↓ due to accumulation of product and ↓ in sub. Factors Affecting Enzymatic Activity • 2- Substrate conc.: • V ά to S till certain point at which the reaction rate is called the maximum rate( V max ) and the active sites on the enzymes are saturated with substrate. • At point B, half of the enzymes are filled with sub. The sub concentration which produce V ½ is known as Km value MichaelisMenten constant ( Km ). Michaelis Constant (Km ) The substrate concentration that produces half the maximal velocity (Vmax/2) is known as Michaelis constant (Km ) Temperature • 1. Increase of velocity with temperature: The reaction velocity increases with temperature until a peak velocity is reached. • This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. • 2. Decrease of velocity with higher temperature: Further elevation of the temperature causes a decrease in reaction velocity as a result of temperature- induced denaturation of the enzyme. • The optimum temperature for most human enzymes is between 35°C and 40°C. Human enzymes start to denature at temperatures above 40°C. PH • The pH at which maximal enzyme activity is achieved is different for different enzymes and often reflects the [H+] at which the enzyme functions in the body. • For example:pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment. • Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains. Inhibitors • Several factors can limit enzyme activity levels, including: • Competitive inhibitors: This inhibitor molecule blocks the active site so that the substrate has to compete with the inhibitor to attach to the enzyme. • Non-competitive inhibitors: This molecule binds to an enzyme somewhere other than the active site and reduces how effectively it works. • Uncompetitive inhibitors: This inhibitor binds to the enzyme and substrate. The products leave the active site less easily, which slows the reaction. • Irreversible inhibitors: This is an irreversible inhibitor, which binds to an enzyme and permanently inactivates it. • Proenzyme (zymogen) – inactive form of enzyme that becomes active after partial proteolysis • example: pepsinogen → pepsin Enzyme definitions • Enzyme :(simple) Protein only enzyme that facilitates a chemical reaction • Coenzyme: Compound derived from a vitamin (e.g. NAD+ ) that assists an enzyme in facilitating a chemical reaction • Cofactor: Metal ion (e.g. Mg2+) that that assists an enzyme in facilitating a chemical reaction • Apoenzyme: Protein only part of an enzyme (e.g. isocitrate dehydrogenase) that requires an additional coenzyme to facilitate a chemical reaction (not functional alone) • Holoenzyme: Combination of the apoenzyme and coenzyme which together facilitating a chemical reaction. • Proenzyme (zymogen) – inactive form of enzyme that becomes active after partial proteolysis • example: pepsinogen → pepsin ENZYMES IN CLINICAL DIAGNOSIS • Some enzymes show relatively high activity in only one or a few tissues. The presence of increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue. • For example, the enzyme alanine aminotransferase ([ALT] is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. • Note: Measurement of ALT is part of the liver function test Isoenzymes • isozymes (also known as isoenzymes or more generally as multiple forms of enzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. • Quaternary structure of isoenzymes: Many isoenzymes contain different subunits in various combinations. • For example: Isoenzymes • 1- The enzyme lactate dehydrogenase is a tetramer made of two different sub-units, the H-form and the M-form. These combine in different combinations depending on the tissue. Type Composition Location Whether destroyed byHeat (at 60 °C) Electrophoretic Mobility Percentage of normalserum in humans LDH1 HHHH Heart and Erythrocyte Fastest No 25% LDH2 HHHM Heart and Erythrocyte Faster No 35% LDH3 HHMM Brain and Kidney Fast Partially 27% LDH4 HMMM Skeletal Muscle and Liver Slow Yes 8% LDH5 MMMM Skeletal Muscle and Liver Slowest Yes 5% 2- CK ( creatine kinase) occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: Isoenzyme Subunit Tissue of Origin CPK1 BB Brain CPK2 MB Heart CPK3 MM Skeletal muscle Isoenzymes – General Features • Genetically determined differences in primary structure • Catalyze the same reaction • May have different subcellular distribution (cytoplasm × mitochondria) • May have different tissue distribution • May be combined from more subunits (quarternary structure) • May differ in kinetic properties (KM) • Usually are determined by electrophoresis 45