Biochem sheet (5) done by: razan krishan corrected by: Shatha Khtoum DATE :4/10/2016

Transcription

1 Biochem sheet (5) done by: razan krishan corrected by: Shatha Khtoum DATE :4/10/2016

2 Note about the last lecture: you must know the classification of enzyme Sequentially. * We know that a substrate binds to active site of enzyme, and enzyme-substrate complex is formed. * Enzyme-substrate complex is transformed to a new product with free enzyme (enzyme is reusable) or may dissociate in to substrate and enzyme ( that means the enzyme first combines reversibly with its substrate ).6:17 First-order kinetics (rate depends on substrate concentration) Zero-order kinetics (rate does not depend on substrate concentration) * It can be observed from the graph that as the concentration of the substrate increases, there is a corresponding increase in the V0. However, beyond a particular substrate concentration, the velocity remains constant without any further increase. This maximum velocity of an enzyme catalyzed reaction under substrate saturation is called the Vmax, Maximum velocity. What does Michaelis constant Km means? 8:50 is the substrate concentration at which the reaction rate is half of Vmax. * equations in slids ( 7+8+9) doctor just read them. the importance of Km is illustrated in this example :

3 The physiological consequence of KM is illustrated by the sensitivity of some individuals to ethanol. Such persons exhibit facial flushing and rapid heart rate (tachycardia) after ingesting even small amounts of alcohol. In the liver, alcohol dehydrogenase converts ethanol into acetaldehyde Normally, the acetaldehyde, which is the cause of the symptoms when present at high concentrations, is processed to acetate by acetaldehyde dehydrogenase. Most people have two forms of the acetaldehyde dehydrogenase, a low KM mitochondrial form and a high KM cytosolic form. In susceptible persons, the mitochondrial enzyme is less active due to the substitution of a single amino acid, and acetaldehyde is processed only by the cytosolic enzyme. Because this enzyme has a high KM, less acetaldehyde is converted into acetate; excess acetaldehyde escapes into the blood and accounts for the physiological effects. Lineweaver-Burk plot. 12:05 We can't use Micheal-Menten model to describe the relation between substrate concentration and initial velocity of Allosteric enzyme Lineweaver and Burk found model to describe it by converting hyperbola curve to straight line. *Lineweaver-Burk polt also known as the double reciprocal plot can be used to calculate Km and Vmax as well as determine the mechanism of action of enzyme inhibitors. In this plot we see that 1/vo is plotted versus 1/[S] and a straight line is obtained.

4 The graph shows that the intercept on the x-axis is equal to -1/Km and the intercept on the y-axis is equal to 1/Vmax. *Turnover number.13:30 Turnover number (kcat): is the moles of S converted to P per mole of E per second. Unit of kcat is mol S*mol E -1 *s -1 The higher Kcat value the higher the reactivity of the E Less activity of enzyme Enzyme inhibition : we two types of enzyme inhibitor. 15:23min 1-Reversible inhibitor : a substance that binds to an enzyme to inhibit it, but can be released -competitive inhibitor :the inhibitor competes with substrate for the active site. ( they're similar in a part of structure.that bind to active site ) and the inhibitor can be relieved by increasing substrate concentration. Example : Methotrexate (inhibitor) which uses in cancer treatment. is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase. It bind to dihydrofolate reductase and inhibits nucleotide base synthesis. -noncompetitive inhibitor : there are two binding sites : active site and allosteric site. the inhibitor binds to allosteric site and inhibit enzyme. the substrate concentration doesn't affect the inhibitor

5 * Competitive and noncompetitive inhibition illustrated on doublereciprocal plot. Competitive inhibition The graph illustrates that the inhibitor has no effect on Vmax but increase Km Noncompetitive inhibitor The graph illustrates that the inhibitor has no effect Km and decrease Vmax 2-Irreversible inhibitor :causes complete inhibition that cannot be reversed such as cyanides and penicillin.( there are three example of irreversible inhibitor they are important. slids )

6 Enzyme regulation. 23:41 min* Enzyme activity can be regulated by several ways: 1-covalent modification (phosphorlation) Some enzymes are active when they're phosphorylated and other when they're dephosphorylated. To phosphorylate enzyme we use protein kinases And to remove phosphate groups we use protein phosphatases. 2-noncovalent modification Using allosteric effectors (activator and allosteric inhibitor ) When they are high levels of AMP that means the energy is depleted and glycogen phosphorylase is active 3-control amount of enzyme : enzyme synthesis: gene regulation enzyme degradation 4-zymogen :is an inactive form of enzyme and is stored in golgi complex. cleavage of one or more covalent bonds transforms it into the active enzyme Example : Chymotrypsinogen a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds trypsin cleaves chmotrypsinogen to π-chymotrypsin (active) contains 15 amino acid then The activated π-chymotrypsin reacts with other π-chymotrypsin molecules to cleave out two dipeptides and yields α- chymotrypsin (active). but doctor said that π-chymotrypsin catalyses itself by cleave out two dipeptided.

7 Trypsingen is transformed to tripsin which cleaves zymogens ( red ones) to their active forms (yellow) The same idea in blood-clotting cascade there are 9 zymogens (inactive enzymes ) which are cleaved by other enzyme. Many enzymes require cofactors for activity.36:06 min Cofactor* Apoenyme : protein part Cofactor: is a non-protein chemical compound or metallic ion that is required for a protein's biological activity to happen. Cofactor can be coenzyme ( water- soluble vitamins). And these vitamins have no toxicity effects. (table 7.1 of coenzyme. you must know coenzyme. their reaction and their vitamin precursor). isoenzymes. 41:40 min* are enzymes that differ in amino acid sequence yet catalyze the same reaction. Usually, these enzymes display different kinetic parameters, such as K M, or different regulatory properties. example : iosenymes of lactate dehydrogenase if there four subunits of H. found in the heart. if there four subunits of M. found in skeletal muscle. pyruvate inhibits H4 so there is no lactate accumulation in the heart. But pyruvate doesn't inhibit M4 so skeletal muscle exposed to spams. In clinical diagnosis we must know the total isoenzymes, and the concentration of each one.