Glycolysis. Biochemistry of Metabolism. glucose-6-phosphate. ATP adenosine triphosphate

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1 Biochemistry of Metabolism opyright by Joyce J. Diwan. All rights reserved. Gibbs Free Energy hanges Rxn# Enzyme ΔG '(kj/mol) ΔG(kJ/mol) exokinase Phosphogluco-isomerase Phosphofructokinase Aldolase Triose phos. Isomerase G--PD Phosphoglycerate kinase Phosphoglycerate mutas Enolase Pyruvate kinase Identify: endergonic rxns exergonic rxns coupled reactions oxidation/reduction rxns Page transfer reactions P -- takes place in the cytosol of cells. Glucose enters the pathway by conversion to --. Initially there is energy input corresponding to cleavage of two ~P bonds of. P Mg + exokinase --. exokinase catalyzes: Glucose + --P + ADP The reaction involves nucleophilic attack of the hydroxyl of on P of the terminal of. binds to the enzyme as a complex with Mg ++. Page Page adenosine tri P P P Mg ++ interacts with negatively charged oxygen atoms, providing charge compensation & promoting a favorable conformation of at the active site of the exokinase enzyme. ribose adenine P Mg + exokinase -- The reaction catalyzed by exokinase is highly spontaneous. A phosphoanhydride bond of (~P) is cleaved. The ester formed in -- has a lower ΔG of hydrolysis. Page Page

2 Induced fit: Glucose binding to exokinase stabilizes a conformation in which: P Mg + exokinase -- exokinase the hydroxyl of the bound is close to the terminal of, promoting catalysis. exokinase water is excluded from the active site. This prevents the enzyme from catalyzing hydrolysis, rather than transfer of to Page 7. It is a common motif for an enzyme active site to be located at an interface between protein domains that are connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, as substrate binding promotes a particular conformation. Page 8. Phospho Isomerase catalyzes: --P (aldose) fructose--p (ketose) The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure. A similar reaction catalyzed by Triose Isomerase will be presented in detail. Page 9 P P Phospho Isomerase -- fructose-- Phosphofructokinase P P P Mg + fructose-- fructose-,-bis. Phosphofructokinase catalyzes: fructose--p + fructose-,-bisp + ADP This highly spontaneous reaction has a mechanism similar to that of exokinase. The Phosphofructokinase reaction is the rate-limiting step of. Page The 0 enzyme is highly regulated, as will be discussed later. P P fructose-,- bis Aldolase P + dihydroxyacetone P glyceraldehyde-- Triose Isomerase lysine + + P + ( ) P Enzyme Schiff base intermediate of Aldolase reaction. Aldolase catalyzes: fructose-,-bis dihydroxyacetone-p + glyceraldehyde--p The reaction is an aldol cleavage, the reverse of an aldol condensation. ote that atoms are renumbered in products of Aldolase. Page A lysine residue at the active site functions in catalysis. The keto group of fructose-,-bis reacts with the ε-amino group of the active site lysine, to form a protonated Schiff base intermediate. leavage of the bond between & follows. Page

3 P P fructose-,- bis Aldolase P + dihydroxyacetone P glyceraldehyde-- Triose Isomerase Triose Isomerase P P P dihydroxyacetone enediol glyceraldehyde intermediate -. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-p glyceraldehyde--p continues from glyceraldehyde--p. TIM's K eq favors dihydroxyacetone-p. Removal of glyceraldehyde--p by a subsequent spontaneous reaction allows throughput. Page The ketose/aldose conversion involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phospho Isomerase. Active site Glu and is residues are thought to extract and donate protons during catalysis. Page P P proposed phosphoglycolate enediolate transition state intermediate analog -Phosphoglycolate is a transition state analog that binds tightly at the active site of Triose Phosphate Isomerase (TIM). This inhibitor of catalysis by TIM is similar in structure to the proposed enediolate intermediate. TIM is judged a "perfect enzyme." Reaction rate is limited Triose Isomerase structure is an αβ barrel, or TIM barrel. In an αβ barrel there are 8 parallel β-strands surrounded by 8 α-helices. Short loops connect alternating β-strands & α-helices. TIM only by the rate that substrate collides with the enzyme. Page Page TIM barrels serve as scaffolds for active site residues in a diverse array of enzymes. Residues of the active site are always at the same end of the barrel, on -terminal ends of β-strands & loops connecting these to α-helices. Page 7 TIM There is debate whether the many different enzymes with TIM barrel structures are evolutionarily related. In spite of the structural similarities there is tremendous diversity in catalytic functions of these enzymes and little sequence homology. Page of 8 the substrate. TIM P P proposed phosphoglycolate enediolate transition state intermediate analog Explore the structure of the Triose Isomerase (TIM) homodimer, with the transition state inhibitor -phosphoglycolate bound to one of the TIM monomers. ote the structure of the TIM barrel, and the loop that forms a lid that closes over the active site after binding

4 Glyceraldehyde-- Dehydrogenase + + P AD + AD + P i P P glyceraldehyde- -. Glyceraldehyde-- Dehydrogenase catalyzes: glyceraldehyde--p + AD + + P i Glyceraldehyde-- Dehydrogenase + + P AD + AD + P i P P glyceraldehyde- - Exergonic oxidation of the aldehyde in glyceraldehyde- -, to a carboxylic acid, drives formation of an acyl, a "high energy" bond (~P). This is the only step in in which AD + is reduced to AD. Page 9,-bisphosphoglycerate + AD + + Page 0 + S cysteine,-bisphosphoglycerate,-bisphosphoglycerate P glyceraldehyde-- A cysteine thiol at the active site of Glyceraldehyde-- Dehydrogenase has a role in catalysis. The aldehyde of glyceraldehyde-- reacts with the cysteine thiol to form a thiohemiacetal intermediate. xidation to a carboxylic acid (in a ~ thioester) occurs, as AD + is reduced to AD. Enz-ys S P glyceraldehyde-- Enz-ys S P AD + AD thiohemiacetal intermediate Enz-ys S P P i acyl-thioester intermediate Enz-ys S P P,-bisphosphoglycerate The high energy acyl thioester is attacked by P i to yield the acyl (~P) product. Page Page Page + R AD + Recall that AD + accepts e plus one + (a hydride) in going to its reduced form. e + + R AD Phosphoglycerate Kinase P ADP Mg + P P,-bisphosphoglycerate -phosphoglycerate 7. Phosphoglycerate Kinase catalyzes:,-bisphosphoglycerate + ADP -phosphoglycerate + This transfer is reversible (low ΔG), since one ~P bond is cleaved & another synthesized. The enzyme undergoes substrate-induced conformational Page change similar to that of exokinase.

5 Page Phosphoglycerate Mutase P -phosphoglycerate P -phosphoglycerate 8. Phosphoglycerate Mutase catalyzes: -phosphoglycerate -phosphoglycerate Phosphate is shifted from the on to the on. Page Phosphoglycerate Mutase P -phosphoglycerate P -phosphoglycerate An active site histidine side-chain participates in P i transfer, by donating & accepting. The process involves a,-bis intermediate. View an animation of the Phosphoglycerate Mutase reaction. histidine + + P P,-bisphosphoglycerate Enolase + P P P -phosphoglycerate enolate intermediate phosphoenolpyruvate 9. Enolase catalyzes: -phosphoglycerate phosphoenolpyruvate + This dehydration reaction is Mg ++ -dependent. Mg ++ ions interact with oxygen atoms of the substrate carboxyl group at the active site. The Mg ++ ions help to stabilize the enolate anion intermediate that forms when a Lys extracts + from #. Page 7 Page 8 ADP P phosphoenolpyruvate pyruvate 0. catalyzes: phosphoenolpyruvate + ADP pyruvate + This transfer from PEP to ADP is spontaneous. PEP has a larger ΔG of hydrolysis than. Removal of P i from PEP yields an unstable enol, which spontaneously converts to the keto form of pyruvate. Required inorganic cations K + and Mg ++ bind to anionic residues at the active site of. Page 9 ADP P phosphoenolpyruvate enolpyruvate pyruvate Page 0 exokinase ADP -- fructose-- Phosphofructokinase ADP fructose-,-bis Phospho Isomerase Aldolase glyceraldehyde-- + dihydroxyacetone- Triose Isomerase continued

6 continued. Recall that there are GAP per. Page glyceraldehyde-- AD + + P i Glyceraldehyde-- AD + + Dehydrogenase,-bisphosphoglycerate ADP -phosphoglycerate Phosphoglycerate Kinase Phosphoglycerate Mutase -phosphoglycerate Enolase phosphoenolpyruvate ADP pyruvate Page Balance sheet for ~P bonds of : ow many ~P bonds expended? ow many ~P bonds of produced? (Remember there are two fragments from.) et production of ~P bonds of per : Balance sheet for ~P bonds of : expended produced ( from each of two fragments from ) et production of ~P bonds of per. - total pathway, omitting + : + AD + + ADP + P i pyruvate + AD + In aerobic organisms: pyruvate produced in is oxidized to via Krebs ycle AD produced in & Krebs ycle is reoxidized via the respiratory chain, with production of much additional. Page Fermentation: Anaerobic organisms lack a respiratory chain. Glyceraldehyde-- Dehydrogenase + + P AD + AD + P i P P glyceraldehyde- -,-bisphosphoglycerate They must reoxidize AD produced in through some other reaction, because AD + is needed for the Glyceraldehyde-- Dehydrogenase reaction. Usually AD is reoxidized as pyruvate is converted to a more reduced compound. The complete pathway, including and the Page reoxidation of AD, is called fermentation. Lactate Dehydrogenase AD + + AD + Lactate Dehydrogenase AD + + AD + pyruvate lactate pyruvate lactate E.g., Lactate Dehydrogenase catalyzes reduction of the keto in pyruvate to a hydroxyl, yielding lactate, as AD is oxidized to AD +. Lactate, in addition to being an end-product of fermentation, serves as a mobile form of nutrient energy, & possibly as a signal molecule in mammalian organisms. ell membranes contain carrier proteins that facilitate transport of lactate. Page Page Skeletal muscles ferment to lactate during exercise, when the exertion is brief and intense. Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs ycle or (in liver) converted to back to via gluconeogenesis

7 Page 7 Lactate Dehydrogenase AD + + AD + pyruvate lactate Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment to lactate and release it. Lactate taken up by adjacent neurons is converted to pyruvate that is oxidized via Krebs ycle. Page 8 Pyruvate Alcohol Decarboxylase Dehydrogenase AD + + AD + pyruvate acetaldehyde ethanol Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product. AD is converted to AD + in the reaction catalyzed by Alcohol Dehydrogenase. Enzyme/Reaction exokinase ΔG o ' kj/mol -0.9 ΔG kj/mol -7., omitting + : + AD + + ADP + P i pyruvate + AD + Fermentation, from to lactate: + ADP + P i lactate + Anaerobic catabolism of yields only high energy bonds of. Phospho Isomerase Phosphofructokinase Aldolase Triose Isomerase Glyceraldehyde--P Dehydrogenase & Phosphoglycerate Kinase Phosphoglycerate Mutase negative Enolase Page 9 Page 0 *Values in this table from D. Voet & J. G. Voet (00) Biochemistry, rd Edition, John Wiley & Sons, ew York, p.. Flux through the pathway is regulated by control of enzymes that catalyze spontaneous reactions: exokinase, Phosphofructokinase &. Local control of metabolism involves regulatory effects of varied concentrations of pathway substrates or intermediates, to benefit the cell. Global control is for the benefit of the whole organism, & often involves hormone-activated signal cascades. Liver cells have major roles in metabolism, including maintaining blood levels various of nutrients such as. Thus global control especially involves liver. Some aspects of global control by hormoneactivated signal cascades will be discussed later. Page exokinase is inhibited by product --: by competition at the active site by allosteric interaction at a separate enzyme site. ells trap by phosphorylating it, preventing exit on carriers. Product inhibition of exokinase ensures that cells will not continue to accumulate from the blood, if [--] within the cell is ample. Page P Mg + exokinase -- 7

8 Glucokinase is a variant of exokinase found in liver. P Mg + exokinase -- Glucokinase has a high K M for. It is active only at high []. ne effect of insulin, a hormone produced when blood is high, is activation in liver of transcription of the gene that encodes the Glucokinase enzyme. Glucokinase is not subject to product inhibition by --. Liver will take up & phosphorylate even when liver [--] is high. Glucokinase is subject to inhibition by glucokinase regulatory protein (GKRP). The ratio of Glucokinase to GKRP in liver changes in different metabolic states, providing a mechanism for modulating phosphorylation. Page Page Glycogen Glucokinase, with high K M for, allows liver to store as glycogen in the fed state when blood [] is high. Glucose exokinase or Glucokinase Glucose--Pase Glucose--P Glucose--P Glucose + P i Pathway Pyruvate Glucose metabolism in liver. Glucose--phosphatase catalyzes hydrolytic release of P i from --P. Thus is released from the liver to the blood as needed to maintain blood []. The enzymes Glucokinase & Glucose--phosphatase, both found in liver but not in most other body cells, allow the liver to control blood []., the last step, is controlled in liver partly by modulation of the amount of enzyme. ADP P phosphoenolpyruvate pyruvate igh [] within liver cells causes a transcription factor carbohydrate responsive element binding protein (hrebp) to be transferred into the nucleus, where it activates transcription of the gene for. This facilitates converting excess to pyruvate, which is metabolized to acetyl-oa, the main precursor for synthesis of fatty acids, for long term energy storage. Page Page P Phosphofructokinase P P Mg + fructose-- fructose-,-bis PFK Activity low [] high [] Phosphofructokinase is usually the rate-limiting step of the pathway. Phosphofructokinase is allosterically inhibited by. At low concentration, the substrate binds only at the active site. At high concentration, binds also at a lowaffinity regulatory site, promoting the tense conformation [Fructose--] mm The tense conformation of PFK, at high [], has lower affinity for the other substrate, fructose--p. Sigmoidal dependence of reaction rate on [fructose--p] is seen. AMP, present at significant levels only when there is extensive hydrolysis, antagonizes effects of high. Page 7 Page 8 8

9 Glycogen Glucose exokinase or Glucokinase Glucose--Pase Glucose--P Glucose--P Glucose + P i Pathway Pyruvate Glucose metabolism in liver. Inhibition of the enzyme Phosphofructokinase when [] is high prevents breakdown of in a pathway whose main role is to make. It is more useful to the cell to store as glycogen when is plentiful. Page 9 9