Enzyme Activity & Inhibition PDF

# **Objectives** - Factors affecting enzyme activity - Temperature - pH - Substrate concentration - Enzyme concentration - Metals - Regulation of enzyme activity - Inhibition - Allosterically - Feedback inhibition # **What Affects Enzyme Activity?** - Three factors...

# **Objectives** - Factors affecting enzyme activity - Temperature - pH - Substrate concentration - Enzyme concentration - Metals - Regulation of enzyme activity - Inhibition - Allosterically - Feedback inhibition # **What Affects Enzyme Activity?** - Three factors: 1. Environmental conditions 2. Cofactors and Coenzymes 3. Enzyme inhibitors # **Factors Affecting Enzyme Activity** 1. Temperature 2. pH 3. Substrate concentration 4. Enzyme concentration 5. Activators or inhibitors | Factors Affecting Enzyme Activity | Rate of Catalysis | |---|---| | Temperature | | | pH | | | Substrate concentration | | | Enzyme concentration | | | Activators or inhibitors | | # **2 Major Factors Affecting Enzyme Rate** - Same factors that cause denaturation... - Temperature - Increase temp. will move molecules faster - pH - Change in H+ concentration will disrupt H-bonds + affect cross-linking - but...break H-bonds in enzyme: not functional # **Factors Affecting Enzyme Function** - Temperature - Optimum T°: - greatest number of molecular collisions - human enzymes = 35°-40°C - body temp = 37°C - Heat: increase beyond optimum T° - increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate - H, ionic = weak bonds - denaturation = lose 3D shape (3° structure) - Cold: decrease T° - molecules move slower - decrease collisions between enzyme & substrate # **pH** - Enzymes also have optimal pHs. These will vary according to the location in which they function. | Location | Optimal pH | |---|---| | Pepsin | 1.4-2 | | Trypsin | 7.7-8 | # **3 Effect of pH on Enzyme Activity** - Each enzyme is active only in a narrow pH range and each has a pH optimum which is often between pH 5.5 and 7.5 (Table). - The optimum pH is affected by the type and ionic strength of the buffer used in the assay. # **pH Optima of Various Enzymes** | Enzyme | Optimal pH | |---|---| | | | # **Effect of Substrate Concentration** - Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. - A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration. - Three distinct phases of the reaction are observed in the graph (A-linear; B-curve, C-almost unchanged. # **Rate Of Catalysis** # **Rate of Catalysis** X = point of saturation # **Enzyme Concentration** - Enzyme Concentration - Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration, when sufficient substrate is present. - Velocity of reaction is increased proportionately with the concentration of enzyme, provided substrate concentration is unlimited. # **Substrate-Specificity (by Shape)** - Substrate = reactant(s) catalyzed by enzyme. - Substrate binds to enzyme on active site. - Forms enzyme-substrate complex (H-bonds). - Induced fit: enzyme changes shape SLIGHTLY to "cuddle" substrate # **Enzyme Catalysis** Enzyme + Substrate(s) <=> Enzyme-substrate complex <=> Enzyme-Products - Enzyme does NOT change in catalysis (retains shape). - Enzyme is ready to catalyze another substrate after reaction is complete (after products released). # **Effect of Activators** - Some of the enzymes require certain inorganic metallic cations Mn, Zn, Ca, Co, Cu, Na, K, for their optimum activity. - Metals function as activators of enzyme velocity through various mechanisms: combining with the substrate, formation of ES-metal complexes, direct participation in the reaction, and bringing a conformational change in the enzyme. # **Michaelis-Menten Equation** Michaelis-Menten Equation: "It is an equation which describes how reaction velocity varies with substrate concentration." $V_o = \frac{V_{max} [S]}{K_m + [S]}$ Where: - $V_o$ is the initial reaction velocity - $V_{max}$ is the maximum velocity. - $K_m$ is the Michaelis constant = $(K_{-1} + k_2) / K_1.$ - $[S]$ is the substrate concentration. # **Michaelis Constant Has Two Significance** 1. $K_m$ is the concentration of substrate at which half the active site of enzymes are filled, thus $K_m$ provide measure of the substrate concentration required for the reaction to occur. 2. $K_m$ is the measure for the strength of the ES complex or the affinity of enzyme to substrate. - a high $K_m$ indicates weak binding with its substrate. - a low $K_m$ indicates strong affinity or binding to substrate. # **Enzyme Activity & Its Inhibition** 1. Inhibition 2. Allosterically 3. Feedback Inhibition # **Inhibitor Of Enzyme** **Enzyme Inhibitors** - **Reversible** 1. Competitive 2. Non-competitive 3. Uncompetitive - **Irreversible** 1. Active site directed 2. Suicide inhibitors # **Irreversible Inhibition** - The irreversible inhibitors are those that bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme's activity; or, those that form a particularly stable noncovalent association. # **Why Are Irreversible Inhibitors Important?** - Irreversible inhibitors are another useful tool for studying reaction mechanisms. - Formation of a covalent link between an irreversible inhibitor and an enzyme is common. # **Active Site Inhibition** | Inhibitor | Effect | Enzyme Inhibited | Condition | |---|---|---|---| | Aspirin | acetylates serine residue in the active center of cyclo-oxygenase | Cyclo-oxygenase | Non-steroidal anti-inflammatory drug (NSAID) | | Difluro methyl ornithine (DFMO) | Irreversible covalent complex with the co-enzyme. | Ornithine decarboxylase | Trypanosomiasis (sleeping sickness) | # **Suicidal Inhibitors** | Inhibitor | Enzyme Inhibited | Therapeutic use | |---|---|---| | Disulfiram | Aldehyde dehydrogenase | Treatment of Alcoholism | | Cyanide | Cytochrome oxidase | Inhibits respiratory chain | | Fluoride | Enolase | Inhibits Glycosis | | Melathion | Acetylcholine esterase | Organophosphorus insecticide | | Di-isopropyl flucrophosphate | Serine proteases, Acetylcholine esterase | Nerve gas | | BAL - British Anti Levsite | reacts with the SH group enzyme | Antidote for heavy metal poisoning | # **(2) Reversible Inhibition** - Reversible inhibitors - after combining with enzyme (El complex is formed) can rapidly dissociate. - Enzyme is inactive only when bound to inhibitor. - EI complex is held together by weak, noncovalent interaction. - Three basic types of reversible inhibition: - Competitive - Uncompetitive - Noncompetitive # **a) Competitive Inhibitors** - A competitive inhibitor often has structural features similar to those of the substrate whose reactions they inhibit. - This means that a competitive inhibitor and enzyme's substrate are in direct competition for the same binding active site on the enzyme. # **STATINS** | STATINS | Enzyme Inhibited | Condition | |---|---|---| | Atorvastatin | HMG CoA reductase | Decrease plasma cholesterol levels - Antihyperlipidemic agents | | Simvastatin | | | | Allopurinol | Xanthine oxidase | Gout | | Methotrexate | Dihydrofolate reductase | Cancer | | Captopril & Enalapril | Angiotensin converting enzyme | High blood pressure | | Dicoumarol | Vit K-epoxide-reductase | Anti-coagulant | # **Noncompetitive Inhibition** <start_of_image> - An inhibitor binds to a regulatory site on the enzyme separate from the active site. # **Noncompetittive Inhibition** | Inhibitor | Enzyme Inhibited | |---|---| | Heavy metals - Ag²-, Hg²+, Pb²+| Binding with cysteinyl SH gr of E | | Pepstatin | Pepsin | | Soyabean trypsin inhibitor | Trypsin | | Ethanol or narcotic-drugs | Acid phosphatase | # **C. Uncompetitive Inhibition** - Here inhibitor does not have any affinity for free enzyme. - Inhibitor binds to enzyme-substrate complex; but not to the free enzyme. - In such cases both Vmax and Km are decreased (Fig. 5.24). - Inhibition of placental alkaline phosphatase (Reganiso-enzyme) by phenylalanine is an example of uncompetitive inhibition. # **(b) Uncompetitive Inhibition** E + S <=> ES <=> E + P - An uncompetitive inhibitor binds at a site other than the active site and, binds only to the ES complex. # **Examples of Uncompetitive Inhibition** - Drugs to treat cases of poisoning by methanol or ethylene glycol act as uncompetitive inhibitors. - Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are uncompetitive inhibitors of liver alcoholdehydrogenase. # **Allosteric Enzymes** - Allosteric enzymes are those having “other shapes” or conformations induced by the binding of modulators. - These enzymes have two receptor sites. - One site fits the substrate like other enzymes. - The other site fits an inhibitor or activator molecule. - Allesteric Enzymes are very important in feedback regulation. # **Example of Allosteric Enzymes** | Pathway | Enzyme | Inhibitor | Activator | |---|---|---|---| | Glycolysis | Phosphofructokinase-1 | ATP & citrate | AMP | | TCA cycle | Isocitrate dehydrogenase | ATP | ADP | | Glycogenolysis | Glycogen phosphorylase | ATP | AMP | | Gluconeogenesis | Fructose 1,6 bisphosphatase | AMP | ATP & citrate | | Fatty acid synthesis | Pyruvate carboxylase | - | Acetyl CoA | | | Acetyl CoA carboxylase | - | Citrate | # **Feedback Inhibition** - System of enzyme regulation where product of reaction can be signal molecule (inhibition -> repressor) - Competitive feedback inhibition: Product binds directly to active site - Noncompetitive feedback inhibition: Product binds to allosteric site - Allows for self-regulation: - If too much product, stops catalysis (repress) - If too little product, continues catalysis - Binding not permanent (can difuse away). # **Exmaple of Feedback Inhibition** | Inhibitor | Enzyme Inhibited | Pathway | |---|---|---| | Heme | ALA synthase | Heme synthesis | | Cholesterol | HMG CoA reductase | Cholesterol synthesis | | Glucose-6-phosphate | Hexokinase | Glycolysis | | Acyl CoA | Acetyl CoA carboxylase | Fatty acid synthesis | | CTP | Aspartate Transcarbamoylase | Pyrimidine synthesis | # **Questions** - What are the factors affecting enzyme activity? - What is the optimum temperature of the human body when enzymes function properly? - Name the agents which denature the enzyme activity? - What is the difference between Glucokinase and Hexokinase? - How would you classify enzyme inhibition? - Give the example of irreversible inhibitor? (Active site and suicidal inhibition?) - What is the difference between competitive and non-competitive inhibitors? - Give the example of competitive and non-competitive inhibitions? # **Questions** - A rectangular _graph_ is obtained when velocity is plotted against the _substrate concentration_. - Change in _pH_ concentration will disrupt _bonding_. - Optimum pH of Pepsin in stomach is _. - Optimum pH of Trypsin in small intestine is _. - In _enzyme_ concentration, when sufficient substrate is present, rate of catalysis, is directly proportional to the _enzyme_ concentration. - _Metal_ ions are also needed for _enzyme_ activity e.g chloride ion for __. - Km is the concentration of substrate at which _the active site of enzymes are filled_. - A high Km indicates weak binding with its _substrate _. - The irreversible inhibitors are those that bind _covalently_ with enzyme. - Suicidal inhibitor examples are _. - In reversible inhibitors, _enzyme_ inhibitor complex is held together by _weak_ interaction - Statin inhibits the enzyme _HMG CoA reductase_ through _competitive_ inhibition. - Competitive inhibitor: Same enzyme, but different _substrate_. - Noncompetittive inhibitor: Same enzyme, but different _substrate_. - When inhibitor bind to _enzyme-substrate complex_ but not to the free enzyme, it is _noncompetitive_ inhibition.