ENZYME BIOCHEMISTRY (FACTOR OF AFFECTING ENZYME REACTION) MUHAIMIN DANISH SYAFI BIN BADRUL HISHAM (2022874118) MAYANG SARI BINTI ABDUL SAMAD ( 2022874122) NOR ASYIRIN BINTI ZAINUDDIN (2021482726) SYAFIQAHUSNA BINTI SAHAR (2022602236) NUR SYAHIRA BINTI RUSLI (2021621878)
2 ENZYME & SUBSTRATE CONCENTRATION Substrate A substance that be used to make a final product. Substrate Concentrations The amount of substrate presence that can be turned into product. In enzymatic reactions, the rate of the reaction initially rises with an increase in substrate concentration until it reaches a maximum, known as the limiting rate. Beyond this point, further increases in substrate concentration do not lead to significant changes in the reaction rate. At this point, so much substrate is present that essentially all of the enzyme active sites have substrate bound to them. In other words, the enzyme molecules are saturated with substrate. The excess substrate molecules cannot react until the substrate already bound to the enzymes has reacted and been released. a) The graph illustrates how changing substrate concentration influences the reaction rate catalyzed by a constant amount of enzyme. With more enzymes, there is a faster reaction. More enzyme molecules (more active sites) lead to increased formation of enzymesubstrate complexes, enhancing the catalytic reaction. When all the substrate is bound, the reaction stops accelerating because there is nothing left for additional enzymes to bind to. If there is enough substrate present, elevating the enzyme concentration will result in a higher reaction rate for the enzyme. This characteristic can be applied in the assessment of serum enzyme activities for diagnosing diseases. b) This graph shows the effect of enzyme concentration on the reaction rate at a constant level of substrate. Enzyme Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. Note that the enzymes are rarely saturated with substrates under physiological conditions.
TEMPERATURE Denaturation: Excessive heat disrupts the enzyme‘s tertiary and quaternary structures. Loss of Function: Altered shape leads to loss of catalytic activity. Inactivation: Enzyme becomes ineffective and cannot facilitate reactions. HIGH TEMPERATURE (ABOVE OPTIMAL) Increased Kinetic Energy: Higher temperatures enhance molecular motion. Optimal Enzyme Activity: Enzyme and substrate collisions occur frequently, promoting reaction rates. OPTIMAL TEMPERATURE Slower Reaction Rates: Reduced kinetic energy results in fewer successful collisions. Reduced Enzyme Activity: Enzyme efficiency decreases as temperature drops. Cold Inactivation: Some enzymes may become inactive at low temperatures. LOW TEMPERATURE (BELOW OPTIMAL) Figure 3: The effect of temperature on the reaction rate Thermophiles: Enzymes function optimally at high temperatures. Psychrophiles: Enzymes that operate best at lower temperatures. Applications: Polymerase Chain Reaction (PCR) that relies on the temperature sensitivity of DNA polymerase. ADDITIONAL INFO 3
The pH scale measures acidity or alkalinity by indicating the concentration of hydrogen ions or hydroxides in a sample. pH changes cause ionization of amino acids, altering protein shape and damaging their function. Enzymes, as proteins, are sensitive to pH changes. Extreme pH levels can cause a complete loss of activity in most enzymes. The pH value at which the enzyme is most active is called the optimal pH value. pH EFFECTS ON ENZYME ACTIVITY 1. Enzyme Structure & Activity: Enzyme structure is crucial for function, impacting the speed of chemical reactions. 2. pH Impact: pH changes impact enzyme shape by altering the ionization of amino acids, a crucial factor for enzyme function. 3. Amino Acid Ionization: pH affects enzyme acidic (carboxyl) and basic (amine) amino acids by changing their ionization states. 4. Ionic Bond Disruption: Amino acids must stay ionized for enzyme stability. pH changes can break ionic bonds, altering the enzyme's shape and possibly deactivating it. pH EFFECTS ON SUBSTRATES 1. pH & Substrate Interaction: pH affects substrate charge & shape, possibly impeding binding to the enzyme's active site or catalysis. 2. Reversible Changes: Enzymes and substrates can undergo reversible structural changes within a specific pH range. 3. Denaturation Risk: Extreme pH shifts denature enzymes and substrates, preventing recognition & hindering reactions. OPTIMAL pH 1. Optimal pH for Enzymes: Each enzyme has an optimal pH for maximum activity. 2. Environmental Adaptation: Enzymes in various environments (e.g., stomach or blood) function optimally at specific pH levels. 3. Effect of pH Deviation: Deviating from the ideal pH slows down & eventually stops enzyme activity. 4. Active Site & pH: Enzymes have a binding site for substrates, & pH changes can alter its shape. 5. Deactivation & Recovery: Maintaining the right pH restores normal enzyme function, while pH changes can either permanently deactivate/temporarily hinder enzyme activity. High concentration of H+ High concentration of OHFigure 4: pH scale Figure 6: State of denatured enzyme Figure 5: Graph of pH against enzyme activity pH 4
INHIBITORS An enzyme inhibitor disrupts the usual reaction pathway between an enzyme & substrate. Inhibitors slow or stop enzyme activity, reducing the chemical reaction. TYPE OF ENZYME INHIBITOR COMPETITIVE Competitive inhibitors resemble a substrate, competing for the enzyme's active site. While the inhibitor does not undergo the reaction, it hinders the substrate from binding. NONCOMPETITIVE Non-competitive inhibitors can bind to the free enzyme or enzymesubstrate complex, separate from the active site. Excess substrate will not reverse the inhibition because the inhibitor does not resemble the substrate. 5 Figure 7: Competitive inhibitors If the inhibitor is present in relatively large quantities, it will initially block most of the active sites. The binding site is reversible, some substrate molecules will eventually bind to the active site and be converted to a product. Higher substrate concentration can displace a competitive inhibitor from the active site, possibly reversing the inhibition. Figure 8: Reversible inhibitors Non-competitive inhibition involves a molecule binding to a site other than the active site (an allosteric site). The binding of the inhibitor to the allosteric site causes a conformational change to the enzyme’s active site. As a result of this change, the active site and substrate no longer share specificity, meaning the substrate cannot bind. As the inhibitor is not in direct competition with the substrate, increasing substrate levels cannot mitigate the inhibitor’s effect. REVERSIBLE It temporarily reduces enzymatic activity by forming a non-covalent interaction with the enzyme. Reversible inhibitors can be either competitive or noncompetitive. Competitive inhibition happens when a molecule, resembling the substrate, blocks the enzyme's active site, hindering substrate binding. Higher substrate concentration can alleviate the inhibitor's impact. Figure 9: Non-Competitive inhibitors
Competitive Non-Competitive Where does it act? Active site Alternative site Change of Km Increase Unchanged Change of Vmax Unchanged Decreased Feedback inhibition is a biochemical process using noncompetitive inhibitors to control enzyme activity. The final product inhibits the enzyme catalyzing the first step in a series of reactions, regulating the synthesis of amino acids. Irreversible inhibitors permanently deactivate enzymes by forming strong covalent bonds with specific groups at the active site. Their effect cannot be reversed even with excess substrate. Figure 10: Feedback Inhibitors Figure 11: Irreversible Inhibitors FEEDBACK INHIBITORS IRREVERSIBLE INHIBITORS INHIBITORS 6 RATE OF REACTION Figure 12: Rate of Reaction The rate of reaction is influenced by the type of enzyme inhibitor. Competitive inhibitors compete with the substrate for the enzyme's active site. Increasing substrate concentration can overcome them, boosting the reaction rate. Non-competitive inhibitors alter the enzyme's shape, decreasing activity by binding to a site other than the active site. Unlike competitive inhibitors, increasing substrate won't overcome their effect, leading to a persistently low reaction rate. Table 1: Differences between Competitive and Non-Competitive
COFACTOR Cofactor is an inorganic or tiny organic molecule that bind to the enzyme to increase or facilitate their activity. What is cofactor? Accelerating the rate of reaction: Cofactors enable enzymes to catalyze processes at a faster pace, which improves their efficiency. Enabling substrate binding: Cofactors can attach to enzymes and make it easier for substrates to bind, which is necessary for the enzyme to catalyze the reaction. Taking part in the reaction: Certain cofactors, including vitamins, can participate in the chemical reaction by donating or receiving electrons, changing the enzyme's oxidation state in the process. Regulating the activity of enzymes: Cofactors can control the activity of enzymes by acting as allosteric regulators or by modifying their activity. Maintaining enzyme structure: Certain cofactors, such as iron or zinc, can support the preservation of an enzyme's structure, guaranteeing the enzyme's correct functioning. ROLES OF COFACTOR TYPES OF COFACTORS Coenzymes Inorganic metal ions Prosthetic groups Organic compounds often contain vitamin molecules in their structures. After the reaction, coenzymes detach from the enzyme and loosely associate with it. Allow the formation of the enzyme substrate complex by altering the charge in the active site. Coenzymes that attach themselves to the enzyme molecule permanently and stay there until the process is finished. Numerous cofactors will reside in the active site of the enzyme and help the substrate to bind. An inactive enzyme lacking the cofactor is called an apoenzyme, whereas a complete enzyme with a cofactor is called a holoenzyme. Figure 13: Mechanism of Action 7
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