Oxidation of glyceraldehyde 3 phosphate : glyceraldehyde 3 phosphate on carbon1 it s an aldehyde group, which is oxidized and converted to carboxylic group ( aldehyde - becomes carboxylic acid ) this is done by NAD+ which is reduced to NADH what is so special about the catazing enzyme of the reaction? This is enzyme has a distinct feature which is, it doesn t release the product which is glycrate form 3 phosph glycerate unless Pi (( inorganic phosphate ( phosphoric acid or phosphate group ) is added to the carboxylic group )). it s a specific enzyme, the mechanism is that the intermediate which is the thioester ( aldehyde, substrate ) is covalently bound to the enzyme. after the covalent bond, oxidation of aldehyde to carboxylic acid that binds to the enzyme through thioester bond in athioester intermediate can t be broken unless Pi is added to the carboxylic group. Again : ((( substrate is bound to SH group in the enzyme and the bond is thioester in an intermediate, you would think that this bond can be hydrolyzed by water and will release the product but this doesn t happen ( the enzyme doesn t allow water to hydrolyze the product ) inorganic Pi acts to release the product what s the importance of this character?
importance : the product directly has phosphacel group ( bond ) which is a high energy bond we can use this product to produce ATP in the next reaction as it s a high energy compound because it has a thioester intermediate which is not released unless pi is added to it. Note : theoretically the reaction is divided into two steps - oxidation and phosphorylation but the reality is that they actually happen together as one step, or the Pi adding would be very difficult the 2 steps are coupled together, 1st is highly exergonic and the second is highly endergonic as they happen as one step due to the covalent binding in the intermediate. refer to the slides to see the figures : figure on the lift : 1 st step shows energy of oxidation and the oxidized products, also there is a big activation energy to form a the acyl phosphate intermediate which makes the reaction very slow due to the large activation energy. figure on the right : one step reaction, you can see that the oxidation product is not formed the thioester intermediate is directly formed, it makes the reaction easier because the 2 reactions are coupled together Transfer of phosphate group to ADP produces ATP : 1,3 bisphosphoglycerate is a high energy compound ( 10.5) so it s higher than ATP (7.3) so hydrolysis of this compound can produce ATP as Pi is transferred from this compound to ADP. In this step we generate ATP.
What s the enzyme? 3 phosphoglycerate kinase as the DR said ( because the reaction is reversible you can look at either way, because it transfers Pi from ATP to glycerate it s a kinase ). this is called substrate level phosphorylaion not oxidative phosphorylation. - there is a shift of pi group from carbon 3 to carbon 2 ( rearrangement of the molecule ) the product is 2 phosphoglycerate, the enzyme is mutase mutase when a group is transferred from one carbon to the other in the same molecule.( Pi3 Pi2 in the same molecule ) - this can undergo dehydration which can remove an H and OH and form a double bond and produces phosphoenolpyruvate and the reaction is reversible and catalyzed by the enzyme enolase phosphoenolpyruvate is a high energy compound if hydrolyzed it will produce 14kcal which is more than enough to synthesize ATP so Pi is transferred directly to ADP to form ATP. - the last step, transfer of Pi to produce pyruvate : the enzyme is pyruvate kinase, it s an irreversible reaction and it produces lots of energy (-7) note : 2ATP will be produced for each glucose molecule. note why is it irreversible the enol group is very unstable so it
becomes pyruvate which is the end product of glycolysis ) The net reaction : glu +2ADP + 2NAD+ + 2 Pi = 2 ATP + 2pyruvate +2 NADH notice that any compound which is used in one step and produced in another step would cancel out and vice versa. the net reaction shows the ultimate goal of glycolysis : which is to produce ATP and pyruvate. Is Oxegyn needed? yes It is because the amount of glucose that can undergo glycolysis is huge ( unlimited ) and the amount of NADH is limited ( NAD+ must be regenerated to repeat the cycle ) so the most common way of regenerating NAD+ is by oxidative phosphorylation. NAD+ is important for continuation of the cycle but 1 or 10 molecules are sufficient to let 1000 or unlimited number of glucose molecules undergo glycolysis. NAD+ becomes NADH glycolysis stops unless NADH is reoxidized and the most common way is by oxidative phosphorylation which requires oxygen so yes and no if we reoxidizenadh at the expense of pyruvate to form lactate NADHis oxidized by transferring its e to pyruvate to produce lactate, no oxygen is needed but if we don t use oxygen, pyruvate won t be the end product, lactate will be the end product. if we want pyruvate as an end product, then oxygen is needed but lactate o isn t needed. so in short as the DR said yes and no.
if oxygen is not available lactate will be the end product in what tissues this happens? - in RBC (compulsory ) it doesn t have mitochondria but it has lots and lots of oxygen. - Cornea ( it has no blood vessels so no oxygen is available ) -- -- exercising muscles, when heavily rapidly, amount of oxygen becomes a limiting factor - note : the red colour of muscles is from myoglobin - RBC have ( lots of oxygen and mitochondria ) - also in lactobacilli bacteria it converts lactose into lactic acid, making yogurt from milk,at 37C and that s where the sour taste comes from - Also : When lactose becomes lactate this affects protein solubility and gives yogurt its sour taste. the next slide it shows different pathways for pyruvate 1) it can become lactate 2) Acetyl coa by decarboxylation and after that it goes for further oxidation 3) Decarboxylation ( removal of carboxyl group ) and production of acetaldehyde which is reduced to ethanol, this occurs in yeast,( makes bread ) also in the bacteria which makes alcohol or vinegar
Note : In making of alcohol or bread glycolysis happens then pyruvate is transformed into acetaldyde which is reduced to ethanol by the energy derived from NADH. so in short the end product is Ethanol in yeast or in the bacteria that makes alcohol. that s why in baking you put some sugar with yeast in warm water ( fermentation reaction ) occurs and CO2 is produced and it s CO2 that gives bread its texture and properties. this reaction also occurs whenever you make alcohol or viniger ( acetic acid ) it also passes through ethanol stage, that s why in making vinegar, containers shouldn t be closed very tightly due to the production of CO2 which damages the container if it s closed too tightly. but the difference here is that ethanol is further converted to acetic acid by other types of bacteria and in making of vinegar and alcohol. Regulation : the role of glycolisis is production of energy in the form of ATP but also formation of building blocks 1) from pyruvate we can add carboxyl group to it and we ll get oxaloacetate which can be converted to cetrate. 2) pyruvate can be converted to alanine by accepting an amino group. so glycolysis doen t have a role in catabolism only, it has a role in anabolism as well. -as in making of amino acids from glucose it goes through glycolysis. 3) to make fatty acids (fats ) from glucose it goes through Glycolysis. Glycolysis has dual role.
So in regulation of glycolysis we should take into consideration its dual role, it s not only for production of ATP,at some points production of ATP is not needed, but synthesis of building blocks is needed. in glycolysis the irreversible reactions are catalyzed, so the enzymes catalyzing irreversible steps are potential sites of regulation, as the irreversible steps are the ones that undergoes regulation in any metabolic pathway. irreversible steps, 3 1,3,10 1 catalyzed by hexokinase 3.. phosphofructokinase 10 by pyruvate kinase to regulate a reaction the best step to regulate it is at the irreversible steps ( irreversible when it happens, no way to go back ). also regulation should occur at an early step, ( the earlier the better ). - regulation at step 1 is better than at step 10 but the best of all is regulation at step 3, because phosphfructokinase produces a product which is used only in glycolysis as the first step produces glucose 6 phosphate which is used in glycogen synthesis, ribosepentose synthesis, NADPH production. in short phosphfructokinase catalyses the committed step as the Dr said. -types of regulation : -reversible binding of allosteric effectors.
- covalent modifications. - the amount of these enzymes ( regulation of transcription ). allosteric enzymes : it has another site other than the active site which is called an allosteric site, it s involved in regulation, decreases or increases the activity. allosteric enzymes usually have multi subunits enzymes, 2 subunits at least, they maybe maid of 4 subunits or more. binding of the substrate to the enzyme is cooperative, it s hard at first, when the one substrate binds to a subunit ( binding site ) binding of a second substrate to another subunit (binding site )becomes easier. this slide shows a 3D structure of phosphofructokinas : 4 identical subunits. There s a catalytic site. And in the middle between the four subunits there is an allosteric site which binds the allosteric molecules ( reversibly ) when their concentration is high they bind to the allosteric site and when it s low they dissociate. - so the binding of allosteric modifiers is rapid and reversible, it decreases or increases the activity of the enzyme, the binding is not at the catalytic site, it s on the allosteric site, it doesn t have to have any relationship with the structure of the substrate. figures : all allosteric enzymes usually exhibit sigmoidal shape, they don t follow mecalistmin kinatecs, there is no hyperpolic
curve, segmoidal curve, the concentration of the substrate ( is very low, a simple increase in the concentration will result in an increase in the reaction velocity ). in a different segment a small increase in the ( bisphosphate ) (refer to the elides please ) will cause a rapid increase in the velocity of the reaction ) this is in high ATP in low ATP the reaction becomes like the other figure so ATP acts in inhibition of the reaction Normally, if you increase the substrate concentration you increase the reaction rate but even If ATP is a substrate for phosphofructokinase. 1) But in ATP increasing the substrate concentration you decease the reaction rate, because it binds at the allosteric site it decreases the activity of the enzyme. The inhibitory ( regulatory ) effect of ATP is due to binding to a regulatory site distinct from the catalytic site which is the allosteric site. if you add AMP with them it cancels the effect of ATP by facilitating binding or competing for the ATP site binding site. 2) also fall in the PH inhibits phosphofructokinase activity. physiological importance of this : glycolysis is producing acids, pyruvic acid or lactic acid.
if the production of lactate continues, they will lead to decrease in PH (increase in the acidity ) as it could be very harmful to other enzymes. 2)inhibited by cetrate, when it s abundant that means that building blocks are available, ** in short high energy and high building blocks inhibits the enzyme ( obviously by logic as the Dr said, do not memorize ). on the other hand fructose 2,6 bisphosphate is a potent activator of phosphofructokinase (2,6 NOT 1,6 ) note : the product of the enzyme is 1,6 bis phosphate the activator is (2,6 bisphosphate ). note that the two compounds are related to each other So the enzyme is called phosphofructokinase 2 but it s product which is fructose 2,6 bisphosphate activates phospofructokinase 1 whose product is fructose 1,6 bis phosphate. y3ne phosphofuctokinase 1 produses Fructose 1,6 bisphosphate and is stimulated by Fructose 2,6 bis phosphate. phosphofructokinase 2 produces 2,6 bis phosphate. To inhibit this and get glycolysis to its normal status we should inhibit the enzyme 2 and to remove Pi from fructose 2,6 bisphosphate that becomes fructose 6 phosphate back again.
It is reversible phosphorylation, by addind Pi we increase glycolysis by removing Pi we decrease the rate of glycolysis. The pi group is removed by an enzyme called phosphatase and added by kinase. On the left : the effect of increasing substrate concentration on reaction velocity The green and blue figures are with fructose 2,6 bisphosphate, the reaction s velocity increases magnificently. The reaction velocity without fructose 2,6 bisphosphate 0MM and substrate concentration is 1 the velocity is 20 % With 0.1 mm 2,6 90 % With 1 MM 2,6 100 % Note : MM micromolar So fructose 2,6 bisphosphate is really a potent activator. ATP concentration in the reaction without the presence of fructose 2,6 bisphosphate violet : the velocity increases but after that it ( by increasing the ATP too much ) decreases due to binding at the allosteric site at 0.1 and 1 MM the effect of ATP decreases. Note : you can see that the curve becomes constant after a while and that s because of saturation of all the enzymes.
PHOSPHOFRUCTOKINASE ENZYME ( refer to the slides slides ) It is composed of 2 domains, a kinase and a phosphatase, a kinase that adds ATP and a phosphatase that removes ATP, and it has a small regulatory region. but when Pi is added and when its removed? the enzyme itself undergoes reversible phosphorylation. at high glucose condition, ansulin is also high, produced level of camp, reduced level of active protein kinase A which depends on levels of camp and glucagon. High insulin Protein kinase is inactive ( the Dr said sth I didn t get from the record ) decreased protein kinase activity favoured dephosphorylation of the phosphofructokinase removed Pi group active kinase and inactive phosphatase high level of fructose 2,6 bisphosphate activation of phsphofructokinase 1 glycolylisis continues Low glucose high glucan (the opposite of Insulin ) and it will bind to its receptor high camp protein kinase becomes active and adds ATP to the bifunctional enzyme active phosphatase and inactive kinase fructose 2,6 bisphosphate decreases decrease in the formation of fructose 1,6 bisphosphate because of reduced activity of the enzyme phosphfructokinase 1 glycolysis stops.
Note : the enzyme is regulated by phosphrylation and de phosphorylation Done by : Marina Zawaideh - -