Enzymes and Enzyme Regulation MGD-module-S2 Session Three 2022-2023 PDF

MGD-module-S2 Session three 2022-2023 Enzymes and enzyme regulation Dr. Hishyar A. Najeeb, MSc., PhD (UK) Assistant Professor., Clinical Biochemistry & Molecular Medicine References Ø Ø Ø Marks’ Basic Medical Biochemistry Chapters 8, 9, 45 Medical Biochemistry Chapters 5, 6 Lippincott’s Illustr...

MGD-module-S2 Session three 2022-2023 Enzymes and enzyme regulation Dr. Hishyar A. Najeeb, MSc., PhD (UK) Assistant Professor., Clinical Biochemistry & Molecular Medicine References Ø Ø Ø Marks’ Basic Medical Biochemistry Chapters 8, 9, 45 Medical Biochemistry Chapters 5, 6 Lippincott’s Illustrated Reviews: Biochemistry Chapter 5 2 Regulation of Enzyme Activity (Regulatory strategies) 3 Lecture 6 Learning outcomes At the end of this Lecture you should be able to: 6) List the major regulatory mechanisms that control enzyme activity (plus examples). 7) Discuss the allosteric properties of a key regulatory enzyme such as phosphofructokinase. 8) Discuss the concept of enzyme cascades and the use of protein kinases and phosphatases to regulate activity. 9) Define the term zymogen and give examples of enzymes that are derived from zymogens. 10) Explain how activation of the clotting cascade leads to the formation of a fibrin clot. 11) Discuss the mechanisms that are involved in the regulation of clot formation and breakdown 4 Regulation of Enzyme Activity 1. Enzyme quantity – regulation of gene expression (Response time = minutes to hours) a)Transcription b)Translation c)Enzyme turnover 2. Enzyme activity (rapid response time = fraction of seconds) a)Allosteric regulation b)Covalent modification c)Association-disassociation’ d)Proteolytic cleavage of proenzyme 5 Main forms of enzyme regulation 1. Substrate and product concentration 2. Changes in enzyme conformation A. Allosteric control Allosteric enzymes are multi subunit enzymes that contain more than 1 active site for the substrate. Plots of V vs [S] for these enzymes yield sigmoidal rate curves. This is caused by the positive cooperatively – the binding of substrate to one active site enhances substrate binding to the other active sites. 6 The Allosteric site It is a site for fitting of a small molecule whose binding alters the affinity of the catalytic site to the substrate. This small molecule is called allosteric modifier. stimulatory: (making it more fit) increase activity of enzyme, curve shifted to the left. Inhibitory: (making the catalytic site unfit) for binding of the substrate, Decrease activity of enzyme, curve shifted to the right T= (tense) R= (relaxed 7 Phosphofructokinase( PFK) Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP PFK catalyzes 1st committed step in glycolysis (10 steps total) (Glucose + 2ADP + 2 NAD+ + 2Pi 2pyruvate + 2ATP + 2NADH) Phosphoenolpyruvate is an allosteric inhibitor of PFK ADP is an allosteric activator of PFK 8 Vo vs [S] plots give sigmoidal curve for at least one substrate Binding of this allosteric inhibitor or this activator does not effect Vmax, but does alter Km Allosteric enzyme do not follow M-M kinetics 9 B. Covalent modification There are many different types of groups that can be covalently attached to proteins via amino acids e.g. Phosphoryl,, adenyl,, acetyl,, uridyl,, methyl,, palmitoyl,, myristoyl,, ribosyl etc. The most important type of modification for regulation is phosphorylation. Phosphate groups are added to ~OH groups of the amino acids serine,, threonine or tyrosine. The introduction of a bulky, charged group can significantly affect enzyme conformation or substrate binding. Attachment of phosphate group is catalysed by kinase. Phosphorylation is a reversible proccess and removal of phosphate is catalysed by phophatase. 10 11 C. Proteolytic activation For some enzymes inactive protein precursors, known as zymogens, are activated by the removal of part of the polypeptide chain. Many proteases, enzymes that can break peptide bonds, are produced in this form. e.g. blood clotting factors. 12 3. Changes in the amount of enzyme A. Regulation of enzyme synthesis Rate of enzyme synthesis is usually regulated by increasing or decreasing the rate of transcription of mRNA. B. Regulated protein degradation The amount of an enzyme can be regulated by controlling its rate of degradation. Proteins can be tagged for destruction by the addition of a small protein molecule known as ubiquitin. 13 4. Regulation of metabolic pathways i) Feedback inhibition: End product of a pathway inhibits its own rate of synthesis by inhibiting enzymes earlier in the pathway e.g. high [ATP] inhibit catabolic pathways 14 ii) Feedforward activation (i.e) Increased amounts of initial substrate increases the first step in the pathway e.g. high concentrations of ethanol induce microsomal ethanol oxidising enzymes iii) Counter regulation of pathways: (i.e) If a catabolic pathway breaking down compound A is activated then the opposing anabolic pathway making compound A will be inactivated. e.g. glycogenolysis and glycogenesis 15 The blood clotting cascade – an example of a tightly regulated process. 16 Main mechanisms which regulate the blood clotting cascade: 1-Inactive zymogens present at low concentration. Most tissue factors are present as inactive precursors which are present in the blood at very low concentrations which ensures that clotting is not initiated accidentally. 2-Amplification of an initial signal. Damage to blood vessels initiates a cascade of activation resulting in the formation of an insoluble fibrin clot. 3-Feedback activation by thrombin. Activated thrombin enhances the conversion of Factors V, VII and XI to activated forms. 4-Termination of clotting by multiple processes. Clotting is stoped by removal of the activated proteins, proteolytic digestion and the binding of inhbitor molecules. 17 THANKS 18