MgD S3 Lecture 2 PDF

Enzymes and enzyme regulation Main forms of enzyme regulation The Allosteric site • Changes in enzyme conformation It is a site for fitting of a small molecule whose binding alters the affinity of the catalytic site to the substrate. ➢Allosteric control ➢Covalent modification ➢Proteolytic activati...

Enzymes and enzyme regulation Main forms of enzyme regulation The Allosteric site • Changes in enzyme conformation It is a site for fitting of a small molecule whose binding alters the affinity of the catalytic site to the substrate. ➢Allosteric control ➢Covalent modification ➢Proteolytic activation •This small molecule is called allosteric modifier. stimulatory: (making it more fit) increase activity of enzyme, curve shifted to the left. • Control by Substrate and product concentration Inhibitory: (making the catalytic site unfit) for binding of the substrate, Decrease activity of enzyme, curve shifted to the right • Changes in the amount of enzyme ➢Control of enzyme synthesis ➢Control of enzyme degradation 1-Control by Substrate and product concentration Allosteric activator - increases activity of enzyme – curve shifted to the left enzyme act Be [ PIT [ 53T$ . B n n Allosteric inhibitor – decreases activity of enzyme – curve shifted to the right M 2-Changes in enzyme conformation •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 cooperativity – the binding of substrate to one active site enhances substrate binding to the other active sites. •Some of the rate-limiting enzymes in the pathways of fuel oxidation (e.g., muscle glycogen phosphorylase in glycogenolysis, phosphofructokinase-1 in glycolysis, and isocitrate dehydrogenase in the tricarboxylic acid [TCA] cycle) are allosteric enzymes. Other molecules may also bind at sites distinct from the active site to increase or decrease enzyme activity / Covalent modification 3-Changes in the amount of enzyme the allosteric properties of phosphofructokinase. There are many different types of groups that can be covalently attached to proteins via amino acids e.g. phosphoryl, adenyl, acetyl, uridyl, methyl etc. •Regulation of enzyme synthesis PFK-1 is the rate-limiting enzyme of glycolysis and controls the rate of glucose 6-P entry into glycolysis in most tissues. 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 a phosphate group is catalysed by a kinase. Rate of enzyme synthesis is usually regulated by increasing or decreasing the rate of transcription of mRNA. •For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism. • Alterations in enzyme levels as a result of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. • Regulated protein degradation •Phosphorylation is a reversible process and removal of a phosphate group is catalysed by a phosphatase. 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 (a protein that targets proteins for degradation in proteasomes). Allosteric regulation of phosphofructokinase (PFK-1) by: 1. AMP and ATP Under physiologic conditions in the cell, the ATP concentration is usually high enough to inhibit the enzyme. This effect of ATP is opposed by AMP, which binds to activate it. 2. FRUCTOSE 2,6-BISPHOSPHATE Is an allosteric activator of PFK-1 that opposes ATP inhibition. 3. THE CITRATE Is an allosteric inhibitor of PFK-1. Define the term zymogen and give examples of enzymes that are derived from zymogens. Zymogen: the inactive precursor of a proteolytic enzyme. To denote the inactive zymogen form of an enzyme, the name is modified by addition of the suffix “-ogen” or the prefix “pro-.” Examples: Stomach: Pepsinogen (pepsin) Pyruvate dehydrogenase kinase inactivates PDH by phosphorylation with ATP. Reactivation is achieved by the action of pyruvate dehydrogenase phosphatase. 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. This allows the inactive forms to be transported safely to their sites of action without causing premature proteolytic cleavage e.g. blood clotting factors. Regulation of metabolic pathways 1. 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. 2. Feedforward activation: Increased amounts of initial substrate increases the first step in the pathway e.g. certain pathways, such as those involved in the disposal of toxic compounds. 3. Counter regulation of pathways: 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 Pancreas: Trypsinogen (trypsin), Chymotrypsinogen (chemotrypsin), Pro-elastase (elastase), and Procarboxypeptidase (carboxypeptidase) Blood Fibrinogen (fibrin), prothrombin (thrombin), and plasminogen (plasmin). Summary S3 Discuss the concept of enzyme cascades and the use of protein kinases and phosphatases to regulate activity. Enzymatic cascade: is a sequence of successive activation reactions involving enzymes, which is characterized by a series of amplifications stemming from an initial stimulus. Protein kinase: an enzyme that can catalyse the addition of a phosphate group from ATP to Ser/ Thr/Tyr residues in a protein. Protein phosphatase: an enzyme that catalyses the removal of a phosphate group from Ser/Thr/ Tyr residues in a protein. Main mechanisms which regulate the blood clotting cascade: Response of enzyme to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. •For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas the addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen) decreases activity. •Phosphate is a bulky, negatively charged residue that interacts with other nearby amino acid residues of the protein to create a conformational change at the catalytic site. The conformational change makes certain enzymes more active and other enzymes less active. The effect is reversed by the phosphatase that removes the phosphate by hydrolysis. 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, VIII 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. Hemi1n