Glycogenolysis Glycolysis Fermentationv

OUTLINE: Catabolism of Glucose Glycogenolysis phosphorylase debranching enzyme phospho-gluco-mutase (PGM) Glycolysis Introduction & overview; 2 phases Phase I hexokinase phospho-gluco-isomerase (PGI) phospho-fructo-kinase (PFK-1) Aldolase triose-phosphate isomerase (TPI) Phase II glyceraldehyde-3-phosphate dehydrogenase Glucose Utilization Four Major Fates of Glucose Catabolism Anabolism P 1 Glycolysis: Phosphoglucose isomerase (PGI) Setting up Second Phosphorylation • Rationale – Convert C1 from aldehyde to hydroxyl which is easier to phosphorylate • P Slightly thermodynamically unfavorable/reversible – DG°’ = +0.4 kcal/mol – product concentration kept low by pairing with favorable next step to drive reaction forward Glycolysis: Phosphoglucose isomerase (PGI) Mechanism a-D- • The isomerization is catalyzed by the active-site glutamate via general acid/base catalysis. • An aldose (glucose) can isomerize into a ketose (fructose) via an enediol intermediate. 2 Glycolysis: Phosphofructokinase-1 Second Phosphorylation •Rationale – Symmetrical phosphorylation to set up cleavage to 3-carbon sugar •First committed step of glycolysis – fructose 1,6-bisphosphate (Fru 1,6-P2) is committed to become pyruvate and yield energy P •This process uses the energy of ATP. •Highly thermodynamically favorable/irreversible (DG°’ = –3.4 kcal/mol) •Phosphofructokinase-1 is highly regulated. P – by ATP, fructose 2,6-bisphosphate, and other metabolites – do not burn glucose if there is plenty of ATP Glycolysis: Fructose-1,6-bisphosphate Aldolase POCH2CH2 (GAP) • Rationale | OH CH2OP POCH2CH2 CH2OP | OH Aldol Cleavage/ condensation – Six-carbon sugar is cleaved into two three-carbon phosphorylated sugars. • The reverse process is the familiar aldol condensation. • Multiple isoforms of aldolase exist; aldolase A, B, C. P • Multiple mechanisms to result in same product (i.e., convergent evolution): – Animal and plant aldolases employ covalent catalysis. – Fungal and bacterial aldolases employ metal ion catalysis. • Thermodynamically unfavorable/reversible (DG°’ = +5.7 kcal/mol) – product (GAP) concentration kept low to pull reaction forward. P 3 Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Covalent Catalysis (Class I enzymes) Step 1: making the Schiff Base b-D- Acid catalysis H Glu Glu Asp Asp Base Catalysis Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Loss of water produces Schiff base Step 1: making the Schiff Base Glu Glu Asp Asp 4 Base Catalysis Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Critical base catalysis Step 2: C-C bond cleavage Glu Glu Asp Asp Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Step 2: C-C bond cleavage Glu Acid catalysis protonates the carbanion Dihydroxy-acetone Glu Asp Asp Enamine is in resonance with the carbanion. Protonation of the carbanion resolves this to a dihydroxyacetone phosphate (DHAP) triose in Schiff-base attachment to the active-site Lysine. 5 Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Step 2: C-C bond cleavage Glu Product Release is the slow step After product release, protonation state of the acid/base residues returns to starting point. Glu Asp H2O Asp Step 3: Hydrolysis of Schiff base (reverse of Step 1) Glycolysis: Fructose-1,6-bisphosphate aldolase Mechanism Summary Mechanism of Class I Aldolases 6 Glycolysis: Triose-phosphate isomerase (TIM) Original C# in Glucose 1 • Aldolase creates two triose phosphates: 3 2 2 3 1 4 1 5 – glyceraldehyde-3-phosphate (GAP) – an aldose – dihydroxyacetone phosphate (DHAP) – a ketose • • C# in GAP P 6 2 P 3 TIM completes preparatory phase of glycolysis Rationale: – allows convergence to a single chemical pathway (the payoff in phase II) – Need to oxidize C1 of GAP (formerly C3 & C4 of glucose) to carboxylate • • TIM uses same enediol intermediate mechanism as PGI Thermodynamically unfavorable/reversible (DG°’ = +1.8 kcal/mol) – GAP concentration kept low to pull reaction forward Glycolysis: Triose-phosphate isomerase Mechanism pKa ≈14.5 Base catalysis Acid catalysis Base catalysis Acid catalysis TIM caps enediol with loop around Pi so that its not a good leaving group, and the elimination reaction to form methyl glyoxal is limited. 7 Glycolysis: Overview • Two Phases/Four concepts – Preparatory phase • Phosphorylation by ATP • Cleavage – Payoff • Oxidation • Phosphorylation of ADP Glycolysis: Overview • Two Phases/Four concepts – Preparatory phase • Phosphorylation by ATP • Cleavage – Payoff • Oxidation • Phosphorylation of ADP 8 Glycolysis: Glyceraldehyde-3phosphate dehydrogenase (GAPDH) • Rationale: – – – – Recall Pyruvate is an acid; need to oxidize aldehyde incorporates inorganic phosphate generation of a high-energy phosphate compound which allows for net production of ATP via glycolysis! OHP P P • First energy-yielding step in glycolysis • First oxidation: aldehyde to carboxylate (ox)/NAD+ to NADH (red). • Active-site cysteine – forms high-energy thioester intermediate – subject to inactivation by oxidative stress The oxidation of glyceraldehyde is only slightly unfavorable, even coupled to NAD + reduction, but the hydrolysis of bisphosphate is even m ore favorable • Thermodynamically unfavorable/reversible (DG°’ = +1.8 kcal/mol) – coupled to next reaction to pull forward Glycolysis: Glyceraldehyde-3phosphate dehydrogenase (GAPDH) Mechanism Binding Covalent catalysis Product release Oxidation by NAD+ Binding and incorporation of inorganic phosphate(Pi) via phosphorolysis Exchange of co-substrate nucleotide 9 Glycolysis: Phosphoglycerate Kinase (PGK) 1st Production of ATP Phosphoglycerate kinase • Rationale: – substrate-level phosphorylation to make ATP – first of two “payoff” steps • 1,3-bisphosphoglycerate is a high-energy compound. – can donate the phosphate group to ADP to make ATP • Named for the reverse reaction; recall Kinases are enzymes that transfer phosphate groups between ATP and various substrates. • Highly thermodynamically favorable/reversible (DG°’ = –5.5 kcal/mol) – This reaction can pull the entire pathway to this point; but only modestly favorable (–1.9 kcal/mol) Glycolysis: Phosphoglycerate Mutase (PGM) Migration of the Phosphate – OH • Rationale: P – Notice that reduction of C3 and oxidation of C2 means no net redox. – Need to get C3 dehydrated, so need to move phosphoryl group – Need to form high-energy phosphate compound to make glycolysis a net ATP producer. • Mutases catalyze the (apparent) migration of functional groups. • Thermodynamically unfavorable/reversible (DG°’ = +1.1 kcal/mol) – reactant concentration kept high by favorability through PGK reaction. 10 Glycolysis: Phosphoglycerate Mutase (PGM) Mechanism Acid/base Catalysis • Similar to other mutases Base catalysis Acid catalysis • One of the active-site histidines is post-translationally modified to phospho-histidine. • Phospho-histidine donates its phosphate to 3-phosphoglycerate at the C2-oxygen before retrieving the phosphate from the 3-carbon oxygen. – Note that the phosphate from the substrate ends up bound to the same His at the end of the reaction. – Note that the other His acts as an acid/base catalyst 11