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1 Slide # 17 ( michaelis- menten approach) : Most enzymes adopt a certain behavior that involves first order + zero order in their reactions When the German scientist studied the enzyme kinetics, he put an equation in which the substrate concentration is changing, the velocity is also changing and the enzyme activity is affected as a result When you do the experiment described by the equation, you do so many trials, each time you add a different concentration and monitor how much the velocity changes every time you`re changing the concentration and plot every change in the diagram, then you connect them and you have the shape of a hyperbolic graph consisting of two kinetic behaviors first order and a zero order and a bent connecting them together Why do we have the bent? Because it`s a stage in which you have two species of the enzyme. some of them are fully saturated with a substrate and the others are still not fully saturated so you`ll have a mix in between the two states What is the V max? it`s the maximum velocity the enzyme can reach What is K m? concentration of the substrate where 50% of the enzyme`s maximum velocity is obtained.( how much concentration I should have to reach 50% of the enzyme`s maximum velocity ) Please refer to the slides for the equation and the other info Slide # 18 : Lineweaver-Burk double- reciprocal plot *You should know it as a historical term, it s not applicable anymore in science. People no longer work on Lineweaver-burk plots, but it s present in text books for you to understand and know what it is. For instance, it s still used in the comparison between inhibitions which will be seen afterwards. *The michaelis-menten equation results in a hyperbolic curve and so you can t get the enzyme parameters(k m and V max ) nor can you compare between enzymes. You can t know exactly the k m, because the k m of 1 is much different than the k m of 10, and much different than the k m of 5, sometimes it s 5 folds the km of 1 and sometimes it s ten folds, so on a graph of the hyperbolic plot you can t properly separate between the k m values of 1,2 and 3. *So Lineweaver-Burk took the reciprocals of the michaelis- Menten equation (1 over V and 1 over the other side of the equation), and by that turned it into an equation for a straight line(linear equation), where 1/Vmax is the y intercept and Km/V max is the slope. *This was done because it was hard to interpret the values outside the graph. However, now after computers and softwares this has become of no real value. Nowadays you can do an 1Page

2 experiment and measure the velocity while changing the enzyme concentration then you can provide the software with them, and it ll provide you with the parameters k m and V max since it already has the equation. Slide # 19 (significance of K m & V max ) *Turnover number (K cat ) 1-it is how much one mole of enzyme can turn the substrate into one mole of the product. 2-It is the relation between stiochemitry of the enzyme and the product. 3-How many moles of the enzyme are needed to convert the substrate to the product. 4-Turnover number= velocity(v) /enzyme concentration 5- Unit >> (time) -1 time could be second, minute, hour or year. *It is important to know the k cat turnover number for the enzymes because it tells you how much the enzyme is efficient in its work. For example; the enzyme that takes a year to convert the substrate to the product is inefficient. *Reactions in general occur because of the collisions. *The natural collision ratio without any interference from the environment or anything else is 10 8 collision per mole per second. *Enzymes differ in their k cat {how much they can convert the substrate to the product} and that what determines the efficiency of enzymes. *One of the most efficient enzymes in life is the catalase it can speed up the reaction rate to reach k cat of 40 million/second which means it can convert hydrogen peroxide molecules substrate to water and oxygen. *H 2 O 2 hydrogen peroxide is toxic for the cells and it can initiate free radicals so it can cause cancers. So, the catalase is present in all the cells and it has an important function. *It can convert molecules of H 2 O 2 per second to their product and the natural collision rate is 10 8.So, it is almost reaching the maximum collision rate that can occur naturally between the substrate and the enzyme>> it is a very high efficient enzyme. Slide #20 & 21 & 22 : enzyme inhibition, competitive inhibition & noncompetitive inhibition. *Enzymes can be inhibited by different materials than the substrate, by the substrate itself or by the product. *Inhibition = preventing the enzyme from functioning well *Function of the enzyme depends on the conformation of the active site. 2Page

3 *Irreversible inhibition = binding of the material covalently to the active site then it won t be broken down. *Reversible inhibition = binding of the material non covalently to the active site then you can be remove it by different materials & different factors. *Enzyme inhibitors are classified into two categories: 1-competitive: compete with the substrate to bind with the active site. HOW? Because they have similar shapes (slightly different) and geometry, they can bind to the active site, so we are decreasing the no. of the productive collisions between the substrate and the enzyme >> the reaction won't proceed >> no products. *But how to get rid of the effect of this inhibitor? By increasing the substrate concentration in order to increase the chance for substrate to bind to the active site. If that happens it will decrease the velocity of the reaction (but V max is not affected) thus increasing K m, however we can increase it by increasing the concentration of the substrate. E.g.: glucose >>>> glucose 6 phosphate by the hexokinase enzyme this reaction is unfavorable and needs energy through conversion of ATP to ADP. The product in this reaction (glucose-6-phosphate) is a competitive inhibitor to the hexokinase. 2-non competitive: inhibitor that binds to the enzyme in any other place other than the active site, it will change the conformation of the active site slightly affecting the activity (not the binding of the substrate to the enzyme) by changing the geometry of the amino acids in the active site (if we increase the distance between the amino acids by 1 A then it will inhibit the enzyme activity) and it does not affect the K m because it doesn t compete on the active site, however V max is affected because it affects the function of the active site. -In competitive inhibition, by increasing the substrate concentration we can overcome the process of inhibition, so that we can re-reach the V max value ( as shown in the red line). 3Page

4 )Isozymes (isoenzymes) as a mode of regulation): *groups of enzymes that differ slightly in their amino acid sequences. However, they do the same function ( they accelerate the rate of the same reaction) *what is the purpose of the differences in structures? -To get different functions. ( structure-function relationship is the whole scince in biochemistry and other fields of sciences). *if there was a change in amino acids in a place other than the active site, where noncompetitive inhibitors bind, they won't bind to the enzyme. First: hexokinase *It is an enzyme with 4 isomers that have the same function ( converting the glucose to glucose-6-phosphate by using ATP as a source of energy). 1) The isomers 1,2 and 3 are found all over the body except 2 places (liver & pancreas). -are capable of phosphorylating several hexoses such as glucose. - are referred to as "low-k m " isozymes because of a high affinity for glucose even at low concentrations. 2) The isomer 4 ( glucokinase) found in liver & pancreas. -its only hexose substrate is glucose. -its K m for glucose is 100 times higher than that of hexokinases I, II, and III This means that we have to add 100 folds of glucose to reach 50% of velocity of the enzyme. *recall that K m equals the concentration of substrate at which 50% of the enzyme active sites are occupied by substrate. -Glucokinase can only phosphorylate glucose if the concentration of this substrate is high enough. **what is the purpose of phosphorylation of glucose? -The first step in glycolysis is phosphorylation of glucose by a family of enzymes called hexokinases to form glucose 6-phosphate (G6P). This reaction consumes ATP, but it acts to keep the glucose concentration low, promoting continuous transport of glucose into the cell through the plasma membrane transporters. In addition, it blocks the glucose from leaking out - the cell lacks transporters for G6P- (once glucose is phosphorylated it can't go outsid the cell again). Pancreas insulin release: *hexokinase 4 (glucokinase) serves as a glucose sensor to control insulin release. 4Page

5 * Insulin is released from the pancreas in response to rising glucose in the bloodstream. * Insulin works by improving the uptake of glucose from the blood across cell membranes and into the cells of the body, and so takes glucose out of the bloodstream. *when the glucose level drops in the blood >> no more secretion of insulin. Liver glycogen synthesis: *hexokinase 4 (glucokinase) controls the glycogen synthesis in liver. *liver : is the storage of glycogen. -the enzyme musn't be inhibited by the glucose-6-phosphate, if inhibited >> no more glucose enters the cells >> no synthesis of glycogen. Second: Lactate dehydrogenase *it is another form of enzymes that have isoform, there are 5 different isoforms distributed in different tissues inside the body. *this enzyme is needed in places with high metabolic rates and anabolic respiration, such as lungs,kidneys,liver,pancreas...etc. *it has certain concentration in blood. *so if one of the tissues that contain it was broken down "either completely or partially" for any reason, do you expect its concentration to decrease or increase in blood? -increase for sure, because the contents of these damaged tissues will be spreaded extracellularly. **Medical application: -Enzymes' levels in blood are used in Medical diagnosis, and one of the best enzymes used to diagnose heart attacks and Myocardial infarction is :Lactate dehydrogenase. - So if a patient with pain in thorax came to you, you'll make him "ECG" تخطيط قلب -If the result was abnormal then he diffinetly has a problem, but if it was normal you can't make sure that he doesn't have a problem unless you use "cardiac enzymes",such as : lactate dehydrogenase. -this enzyme converts Pyruvate into Lactate and vice versa. 5Page

6 -So, if lactate dehydrogenase levels were high in blood then for sure the patient has a problem that can be either in heart or in liver, but since the patient doesn't have pain in abdomen we can exclude the liver. -to make sure that the problem is in heart, we make " differential diagnosis of lactate dehydrogenase", if the result was " high levels of Lactate dehydrogenase 1 " then for sure the problem is in heart ; some of heart cells were broken down which increased "LACTATE DEHYDROGENASE 1" levels in blood. *little notes:.-it is important to know lactate dehydrogenase, because it is everyday practice in hospitals. f -there are 4 cardiac enzymes that can be used in medical diagnosis.. وال بد من شمس أختي الغالية.. ال بد لنا أن نمأل أوقاتنا بما ينفعنا في ديننا ودنيانا.. فلندع قلوبنا ترتقي معا بحب هللا ولنجعل اإليمان والمحبة جناحين نحلق بهما في سماء أفراحنا... مع أطيب أمنياتنا لكم بالنجاح والتوفيق فريق "" We're sorry for being late Sorry for any mistake 6Page