Biochemistry LC4 Enzymes PDF

COURSE OUTLINE I. INTRODUCTION TO ENZYMES II. ENZYME CLASSIFICATION III. ENZYMATIC REACTION A. Specificity of Enzyme Action B. ES Complex Formation Theories IV. STRUCTURE OF ENZYMES V. FACTORS AFFECTING ENZYME...

COURSE OUTLINE I. INTRODUCTION TO ENZYMES II. ENZYME CLASSIFICATION III. ENZYMATIC REACTION A. Specificity of Enzyme Action B. ES Complex Formation Theories IV. STRUCTURE OF ENZYMES V. FACTORS AFFECTING ENZYME FUNCTION VI. REGULATION OF ENZYME ACTIVITY VII. MICHAELIS-MENTEN EQUATION VIII. MEDICAL AND HEALTH APPLICATIONS IX. REFERENCES ENZYMES I. INTRODUCTION TO ENZYMES ENZYMES Produced by living cells and acts as biological catalysts for many biochemical reactions. ○ All enzymes are proteins except for some that RNAs They are globular proteins with specific three dimensional conformation. CATALYSIS Increases the rate of a chemical reaction without itself being changed or consumed in the overall process. Reaction rate is the decrease in the concentration of reactant per unit of time or the increase in the concentration of product per unit time. II. ENZYME CLASSIFICATION NOMENCLATURE Fig. 1: Six Classes Of Enzymes A. Recommended Name A.1 Most commonly used enzyme names have the suffix “ase” attached to the III. ENZYMATIC REACTIONS substrate of the reaction. Example: Glucosidase and Urease A. SPECIFICITY OF ENZYME ACTION A.2 Description of action performed Example: Lactate dehydrogenase and Steps in an Enzymatic Reaction adenylyl cyclase a. Substrate and enzyme combine to form an *Some enzymes retain their original trivial “enzyme-substrate complex” names like trypsin and pepsin b. The complex undergoes a transition state (not quite the substrate or the product yet) B. Systematic name c. Formation of the product Enzymes are divided into six major classes d. Separation of the product from the enzyme TRANSITION STATE Enzyme- Substrate Complex E + S -> ES -> E+P Held together by weak bonds E is unchanged at the end of reaction, it returns to original shape after releasing P BIOCHEMISTRY LC 4: ENZYMES DR. ESPIRITU, A. DATE: 09/04/2024 ACTIVE SITE The enzyme and substrate must A very specific area on the enzyme which still have complementary recognizes and binds to the substrate surfaces. Contains the binding site and catalytic site Fig. 4: The Induced Fit Model Note: There are also group specific that can accept any number of closely related substances Example: Carboxypeptidase IV. STRUCTURE OF ENZYMES Fig. 2: The Active Site B. ES-COMPLEX FORMATION THEORIES 1. Emil Fischer’s Lock -and-key model (1895) - enzymes and substrates combine because they have complementary molecular geometries - the enzyme is pictures as being conformationally grid - explains the specificity of the enzymes but as structures of enzymes became available, it became clear that not all enzymes have the specific shapes required for the lock-and-key theory Fig. 5 The Structures Of Enzyme COFACTORS Organic or organo-metallic chemicals Fig. 3: the lock and key model Metalloenzymes ○ Zn, Fe Mg, Cu ABSOLUTE Coenzymes - Accepts only one type of ○ NAD, NADP, ATP molecule Prosthetic Group - Can discriminate between D and ○ Co-factors which are covalently L isomers bonded to the enzyme Example: Trypsin 2. Koshland’s Induced-fit hypothesis Other Technical Definitions Related to Enzymes (1958) - the enzyme is a molecule whose Isoenzymes conformation can change as the - enzymes that occur in different molecular substrate approaches and starts forms, but catalyze the same reaction to b ind - proteins are flexible molecules, Ribozymes whose overall structure is - RNA molecules that have catalytic maintained by weak properties intermolecular interactions. - at any given time, these can be Allosteric Enzymes disrupted by small changes in - enzymes that have more than one their vicinity. substrate binding site - The approach of the substrate is - the binding facilities the binding of other viewed as such a perturbation. substrate molecules PREPARED BY: BATCH 2028 1D 2 BIOCHEMISTRY LC 4: ENZYMES DR. ESPIRITU, A. DATE: 09/04/2024 V. FACTORS AFFECTING ENZYME at which it functions best. Deviations from this optimal level can reduce enzyme FUNCTION activity A. SUBSTRATE CONCENTRATION Ionic Interactions: Changes in salinity increase substrate = increase reaction rate can alter the ionic bonds within the more substrate = more frequently collide enzyme or between the enzyme and its with enzyme substrate. This can affect the enzyme’s reaction rate levels off shape and its ability to bind to the all enzymes have active site engaged substrate enzyme is saturated Enzyme Denaturation: High salt B. ENZYME CONCENTRATION concentrations can lead to enzyme denaturation, where the enzyme loses its an increase in enzyme = increase in functional shape. This denaturation can be reaction rate irreversible, leading to a permanent loss of more enzymes = more frequently collide enzyme activity with substrate reaction rate levels off substrate becomes Substrate Availability: Salts can also limiting factor affect the solubility and availability of substrates, further influencing the overall C. TEMPERATURE reaction rat Optimum Temperature higher molecular collisions F. ACTIVATORS - human enzymes =35°- 40° Some of the enzymes require certain inorganic - body temp = 37° metallic cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity. Rarely, - Heat: increase beyond optimum anions are also needed for enzyme activity e.g. Temperature chloride ion (CI–) for amylase. increased energy level of molecules disrupts bonds in enzymes & between G. INHIBITORS enzyme & substrate -H, ionic = weak bonds Compounds that bind to enzymes and reduce their denaturation= lose 3D shape (3° structure) activity. - Cold: decrease Temperature molecules move slower a. Irreversible Inhibitors decrease collisions between enzymes & - inhibitor forms a stable complex with the substrate enzyme (inhibitor does not easily Note: The optimum temperature for most dissociate). Example, heavy metals Pb, human enzymes is between 35°C and Cd, Hg 40°C. Human enzymes start to denature at - removal of bound metals from the enzyme temperatures above 40°C , but can be done by addition of thermophilic bacteria found in the hot chelating agents such EDTA, citrate, etc springs have optimum temperatures of 70°C. b. Reversible Inhibitors - inhibitor may dissociate more easily from D. pH the enzyme after binding - changes in pH adds or remove H+ c. Competitive Inhibition - disrupts bonds, disrupts 3D shape - molecules bind to the active site and - disrupts attractions between charged prevent the substrate from binding amino acids - chemical nature of inhibitor and substrate - affect 2° & 3° structure are similar - denature protein - Km is increased;Vmax is unchanged Optimal pH Most human enzymes: pH 6-8 - depends on localized conditions - pepsin (stomach) = pH 2-3 - trypsin (small intestines) = pH 8 E. SALINITY - Salinity, or salt concentration, can significantly impact enzyme functions in several ways: Optimal Salt Concentration: Each enzyme has an optimal salt concentration Fig. 5: Competitive Inhibition PREPARED BY: BATCH 2028 1D 3 BIOCHEMISTRY LC 4: ENZYMES DR. ESPIRITU, A. DATE: 09/04/2024 Examples: Penicillin - blocks enzyme Examples: bacteria use to build cell walls Lithium - used in the treatment of Disulfiram (Antabuse) - treats bipolar disorder, can act as an chronic alcoholism by blocking uncompetitive inhibitor of inositol enzymes that break down monophosphatase. This inhibition alcohol, as a result, severe affects inositol signaling hangover & vomiting 5-10 pathways, which play a role in minutes after drinking. mood stabilization. d. Non-Competitive Inhibition - molecules that bind to a site other than the VI. REGULATION OF ENZYME ACTIVITY active site but change the - shape of the active site so that it cannot The regulation of the reaction velocity of enzymes is bind the substrate essential if an organism is to coordinate its - conformational change numerous metabolic processes. - Km is unchanged; Vmax is decreased A. REGULATION OF ALLOSTERIC ENZYMES Allosteric enzymes - regulated by molecules called effectors that bind noncovalently at a site other than the active site. a. Homotropic effectors (substrate itself serves as an effector); b. Heterotropic effectors (effector may be different from the substrate) *Note: Effectors can be either positive (accelerate the enzyme catalyzed reaction) or negative (slow down the reaction). Fig 6. Noncompetitive Inhibition Examples: Some anti-cancer drugs- inhibit enzymes involved in DNA synthesis, stop DNA production and division of more cancer cells e. Uncompetitive Inhibition - Binds to regions other that the active site and only to ES complexes - Km is decreased; Vmax is decreased Fig.8: Allosteric Inhibition B. REGULATION OF ENZYMES BY COVALENT MODIFICATION Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. a. Phosphorylation and dephosphorylation - Phosphorylation reactions are catalyzed by a family of enzymes called Fig. 7: Uncompetitive Inhibition PREPARED BY: BATCH 2028 1D 4 BIOCHEMISTRY LC 4: ENZYMES DR. ESPIRITU, A. DATE: 09/04/2024 protein kinases that use ATP as the VIII. MEDICAL AND HEALTH phosphate donor. APPLICATIONS b. Response of enzyme to phosphorylation - Depending on the specific enzyme, the Clinical Diagnosis phosphorylated form may be more or All physiological processes occur in an less active than the unphosphorylated ordered, regulated manner and enzyme. homeostasis is maintained. In a pathologic state, homeostasis can be c. Induction and repression of enzyme profoundly disturbed. The enzyme activity synthesis as reflected in the blood can be a basis for - The increase (induction) or decrease diagnosis and prognosis. (repression) of enzyme synthesis leads Plasma enzymes as diagnostic tools. to an alteration in the total population of Some enzymes show relatively high active sites. activity in only one or a few tissues. The presence of increased levels of these enzymes in plasma thus reflects damage VII. MICHAELIS-MENTEN EQUATION to corresponding tissue. For example, alanine aminotransferase, ALT (also called V=Vmax[S]/(KM+[S]) glutamate-pyruvate transaminase GPT) is abundant in the liver. The increased levels An increased substrate concentration, reaction of ALT in the plasma signals possible approaches its maximum velocity, Vmax (when the damage to hepatic tissues. enzyme is saturated). Diseases of the heart - The plasma levels of creatine kinase (CK), formerly called V max - maximum velocity creatine phosphokinase, CPK), lactate Km - Michaelis constant = [S] at 1/2V max dehydrogenase (LDH),and glutamate Kcat - turnover number oxaloacetate transaminase (GOT) are The maximum # of molecules of substrate commonly determined in the diagnosis of converted to product per active site per unit of time, myocardial infarction.They are particularly =Vmax/[E] useful when the electrocardiogram is difficult to interpret, such as when there have been previous episodes of heart diseases. Pharmacology and Toxicology DNA Technology Reference(s): 1. Dr. A. Espiritu (2024). Lecture and Powerpoint Presentation. Michaelis-Menten graph: Used to visualize enzyme kinetics. With a fixed amount of enzyme, the reaction velocity increases as substrate is added, until the active sites on all of the enzymes become saturated. At this point, the reaction speed plateaus----> vmax Km is found using a Michaelis-Menten diagram by identifying~ Vmax on the y-axis, then finding the corresponding substrate concentration value on the x-axis. PREPARED BY: BATCH 2028 1D 5