MEDF 1012A Biocatalyst Lecture Notes 2024 PDF

MEDF 1012A Foundation Course For Health Sciences II Biocatalyst Dr. Yeung Hang Mee, Po School of Biomedical Sciences Email: [email protected] Office: Choh-Ming Li Basic Medical Sciences Buildin...

MEDF 1012A Foundation Course For Health Sciences II Biocatalyst Dr. Yeung Hang Mee, Po School of Biomedical Sciences Email: [email protected] Office: Choh-Ming Li Basic Medical Sciences Building 6/F Room 610P 1  Learning Objectives Describe the structures and components of enzymes; Understand the enzyme kinetics including Michaelis-Menten equation and Lineweaver-Burk plot; Explain different types of enzyme inhibitions with examples; Review multiple factors affecting enzyme’s activity.  Biocatalyst A catalyst speeds up a chemical reaction compared to the same reaction without the help of catalyst. It is not consumed in the reaction and can be recycled. It increases the rate of a reaction by lowering the activation energy requirement of a reaction. Structures and Components of Enzymes 4  General Properties of Enzymes Most enzymes are proteins: They require cofactors (inorganic) or coenzymes (organic). Some enzymes are ribonucleic acids (RNA) called ribozymes: Ribozymes are capable of catalyzing specific biochemical reactions. They are found in the ribosome where they join amino acids together to form protein chains. Reference: sciencedirect.com  General Properties of Enzymes Enzymes have highly specific reactants called substrates to bind on the active site. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. References: Lippincotts Illustrated Reviews Biochemistry 5th Edition.  General Properties of Enzymes Enzymes operate effectively at specific pH and temperature ranges: Pepsin (stomach) works effectively at very acidic environment. Trypsin (activated at duodenum) works effectively at alkaline environment. Alkaline phosphatase (bones and liver) works effectively at alkaline environment. Most of the enzymes (made by proteins) will be denatured at high temperature due to the change in conformation of its 3D structure. References: Lippincotts Illustrated Reviews Biochemistry 5th Edition & Becker’s World of the Cell 8th Edition.  Cofactors Cofactors are inorganic components that are bound to enzymes for activity. An inactive enzyme without the cofactor is called an apoenzyme. Examples: inorganic metal ions such as zinc, copper, ferric ions.  Coenzymes Coenzymes are organic components that are bound to enzymes for activity. An inactive enzyme without the coenzyme is also called an apoenzyme. Examples: flavin adenine nucleotide (FAD), nicotinamide adenine dinucleotide (NADH).  Classifications of Enzymes Each enzyme is assigned a four-digit classification number and a systematic name according to the reaction catalyzed. References: Biochemistry 7th Edition, W.H. Freeman and Company.  Classifications of Enzymes No. Class Type of reaction catalyzed 1 Oxidoreductase Transfer of electron, hydride ions or hydrogen atom. 2 Transferase Group transfer reaction. 3 Hydrolase Hydrolysis (“hydro” means water). 4 Lyase Addition of groups to double bond, or formation of double bond by removal of group. 5 Isomerase Transfer of groups within molecules to yield isomer. 6 Ligase Formation of C-C, C-S, C-O and C-N bonds by condensation coupled to ATP cleavage. Transfer of a PO32- group References: Biochemistry 7th Edition, W.H. Freeman and Company.  Activation Energy in Uncatalyzed or Catalyzed Reaction Enzyme can provide an alternative pathway with 1 lower activation energy for the chemical reaction. 2 Options: 1? 2? References: Biochemistry 7th Edition, W.H. Freeman and Company. Enzyme Kinetics 13 Michaelis-Menten Equation The Michaelis-Menten equation reflects the change of velocity of enzymatic reaction under different concentrations of substrate provided: Part 1: almost a linear phase since the active sites of all enzymes are not fully occupied. Part 2: a plateau phase since all enzyme’s active sites are fully occupied. Reference: Stryer Biochemistry 6th Edition.  Michaelis-Menten Equation 1. Vmax: the maximum velocity which 1 the enzyme can reach. 2. Km: a specific value of substrate concentration which the velocity of enzymatic reaction reaches to its half Vmax. 2 3. Km value can indicate if the substrate added has a high or low affinity to the particular enzyme. Reference: Stryer Biochemistry 6th Edition.  Michaelis-Menten Equation 1. If glucose is the common substrate for enzyme 1 and enzyme 2: Km1: 2 mM Km2: 7 mM Enzyme 1 2. Which enzyme has a higher affinity Enzyme 2 of glucose? Why? Answer: Enzyme1 has a lower Km value which means a less glucose concentration provided is enough to reach half Vmax Km1 Km2 Glucose concentration [S] (mM) Higher Km value Lower affinity Lower Km value Higher affinity Transformation of Michaelis-Menten Equation into Lineweaver Burk Plot 1 = 1 = + 1 = 1 + Reference: Stryer Biochemistry 6th Edition.  Transformation of Michaelis-Menten Equation into Lineweaver Burk Plot 1 1 = 1 Slope = + For a linear equation: Y axis value = slope * x axis value + y-intercept 1 (y-intercept) 1 Y axis value = 1 -1 (x-intercept) Slope = Y-intercept = 1 X-intercept = -1 Enzyme Inhibitions 19  Types of Enzyme Inhibitions Reversible inhibition: Dissociation of inhibitor from the enzyme or enzyme-substrate complex is rapid (a weaker interaction). Three types: Competitive Non-competitive Un-competitive Irreversible inhibition: Dissociation of inhibitor from the enzyme or enzyme-substrate complex is very slow (a stronger interaction). Also known as “suicide inhibition”, i.e. enzyme activity is permanently inhibited.  Reversibly Competitive Inhibition A competitive inhibitor binds in and competes with substrate for the active site of the enzyme, which results in an apparent increase in KM without changing the Vmax. This happens because the maximal rate of enzymatic reaction (Vmax) can resume if the substrate concentration is increased (a higher KM with a lower affinity). Example: statin for hypercholesterolemia treatment. Vmax still can achieve if you increase S! “他汀類藥物” Reference: Pearson Education 2011.  Reversibly Non-competitive Inhibition A non-competitive inhibitor can bind to another site outside the active site. This leads to lower the Vmax (you cannot resume Vmax by increasing S!) KM of the enzyme is less affected since the substrate still can be bound on the active site, only the product formed is affected. Example: cyanide on cytochrome oxidase. Vmax cannot resume with increasing S! Cyanide “山埃” Reference: Pearson Education 2011.  Reversibly Un-competitive Inhibition An un-competitive inhibitor can bind to another site outside the active site when the enzyme-substrate complex is formed. This leads to lower the Vmax , overall the product will be reduced. KM of the enzyme is reduced since this type of inhibitor favors the formation of enzyme-substrate complex, and the inhibition is more apparent with increasing substrate concentration. Example: monoamine oxidase inhibitor against depression. Vmax and KM are reduced! With inhibitor Reference: Pearson Education 2011.  Enzyme-inhibitor Enzyme-substrate- (ii) Non-competitive type complex inhibitor complex (Target BOTH free enzyme & Inhibitor enzyme-substrate complex) Inhibitor OR Inhibitor Inhibitor Enzyme-inhibitor complex Enzyme-substrate- (i) Competitive type inhibitor complex (Target for FREE enzyme) (iii) Un-competitive type (Target for enzyme-substrate complex ONLY) Types of Enzyme Inhibition by Lineweaver-Burk Plot Normal Non-competitive VMax Competitive Un-competitive KM KM VMax  Irreversibly Inhibition This type of enzyme inhibitor can bind to the active site and form a strong bonding (covalent bond) with the enzyme. Example: Aspirin. The COX is an enzyme which trigger inflammatory responses when: The arachidonic acid (AA), a 20-carbon fatty acid in our body which binds to the active site of COX including the ser- 529. Ser-529 means the serine (amino acid) at the amino acid sequence no. 529. Diagram of Cyclo-oxygenase (COX)  Irreversibly Inhibition This type of enzyme inhibitor can bind to the active site and form a strong bonding (covalent bond) with the enzyme. Example: aspirin The action of aspirin is by: Addition of acetyl group (acetylation) on ser-529. CH3 Factors Affecting Enzyme’s Activity 28  Rate-determining or Rate-limiting Step The metabolic pathway usually involves many steps and the slowest step is defined as rate-determining or rate-limiting. Considering the following examples: Enzyme 1 Enzyme 2 A B C Step 1 Step 2 Reactant A Reactant B Product Ea step 1 Question: which step is the Ea step 2 rate-limiting step? Why? Answer: Step 1 is the rate-limiting step since it has a higher activation energy and it would be slower than step 2  Product Inhibition The product from a series of enzymatic reactions can inhibit the overall reaction rates. Considering the following examples: Enzyme 1 Enzyme 2 A B C Reactant A Reactant B Product Product inhibition prevents waste that occurs when more of a product is made than the cell needs. It can also prevent harm when having too much of the pathway’s end product may actually be harmful to us, e.g. cholesterol synthesis by our liver.  Zymogen Zymogen is an inactive precursor of the enzyme or it is also known as pro-enzyme. The conversion of active enzyme from its zymogen is specific proteolytic cleavage of zymogen. Examples: Inactive pepsinogen -> active pepsin in stomach; Inactive trypsinogen in pancreas-> active trypsin at duodenum. Source: Lehninger Principles of Biochemistry 7th Edition  Premature Activation of Zymogen Premature activation of zymogen is a common phenomenon in acute pancreatitis. In normal conditions, the pancreas produces trypsinogen and secretes into duodenum; the enterokinase (enzyme) in duodenum cleaves the trypsinogen into active trypsin for dietary protein digestion. In acute pancreatitis, the inflammatory response triggers an alternative pathway to activate trypsinogen into trypsin inside the pancreas, and thus the pancreatic tissues are auto-digested by the active trypsin. Source: http://www.scmlifescience.co.kr/en/clinical_test02b.asp  Summary 1. Most of the enzymes are made of proteins and some are made of ribonucleic acids (ribozymes). 2. Cofactors are inorganic components and coenzymes are organic components that are bound to enzymes for activity. 3. There are six different classes for enzyme classifications based on the type of reaction catalyzed. 4. The Michaelis-Menten equation reflects the change of velocity of enzymatic reaction under different concentrations of substrate provided. 5. Dissociation of inhibitor from the enzyme or enzyme-substrate complex is rapid (a weaker interaction) is known as reversible inhibition. 6. Dissociation of inhibitor from the enzyme or enzyme-substrate complex is slow (a stronger interaction) is known as irreversible inhibition. 7. The metabolic pathway usually involves many steps and the slowest step is defined as rate-determining or rate-limiting. 8. Product inhibition prevents waste that occurs when more of a product is made than the cell needs. 9. The conversion of active enzyme from its zymogen (pro-enzyme) is specific proteolytic cleavage of zymogen.