Glycolysis Triose-phosphate Isomerase

OUTLINE: Glycogenolysis phosphorylase – acid/base; carbo-cation; PLP cofactor debranching enzyme phosphoglucomutase - acid/base; phospho-enzyme; bisphosphate int. Glycolysis Introduction & overview; 2 phases Phase I hexokinase - phosphotransferase-coupling PGI - endiol PFK1 - phosphotransferase-coupling Aldolase - Schiff base (electron sink to stabilize a carbanion) TPI - endiol (fast) Phase II GAPDH - oxidation PG kinase - return on investment- substrate-level phosphorylation PG mutase - acid/base phospho-enzyme Enolase - enolate Pyruvate Kinase - phosphotransferase 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. 1 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 2 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 favorable, 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) Exchange of co-substrate nucleotide 3 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. 4 Glycolysis: Phosphoglycerate Mutase (PGM) Mechanism Acid/base Catalysis • Similar to other mutases • One of the active-site histidines is post-translationally modified to phospho-histidine. Base catalysis • Phospho-histidine donates its phosphate to 3-phosphoglycerate at the C2-oxygen before retrieving the phosphate from the 3-carbon oxygen. Acid catalysis – 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 The Mutases Accomplishing Phosphomutase Activity phosphoglycerate mutase • One binding step – 2-phosphoglycerate 3-phosphoglycerate • a-phosphoglucomutase Two binding steps – – – Glycogen metabolism Bis-phosphorylated intermediate flips in “vestibule” No glucose exchange a-glucose 1-phosphate glucose 6-phosphate • b-phosphoglucomutase b-glucose 1-phosphate No reorientation required glucose 6-phosphate Two binding steps – – – – Free bis-phosphorylated intermediate flips outside active site HAD Exchange glucose Micro-organisms 5 Glycolysis: Enolase – OH P •Rationale: – Dehydrates C3 to reduce it like pyruvate – Double-bonded C2-C3 is part of an en-ol except that the C2-alcohol is in ester linkage with a phosphate •2-Phosphoglycerate is not a good enough phosphate donor to generate ATP. – loss of phosphate from 2-PG would give a secondary alcohol, which is completely stable •Slightly thermodynamically unfavorable/reversible (DG°’ = +1.8 kcal/mol) – product concentration kept low to pull forward Glycolysis: Enolase Mechanism Dehydration fast slow – OH P P 6 Glycolysis: Pyruvate Kinase (PK) – Pyruvate kinase P • Rationale: – substrate-level phosphorylation to make ATP – second of two “payoff” steps – net production of 2 ATP/glucose • Phosphoenolpyruvate (PEP) is a high-energy compound. – can donate the phosphate group to ADP to make ATP • Loss of phosphate from PEP yields an enol that tautomerizes into ketone. • Tautomerization – effectively lowers the concentration of the reaction product – drives the reaction toward ATP formation • Named for the reverse reaction; recall Kinases are enzymes that transfer phosphate groups between ATP and various substrates. • Pyruvate kinase requires divalent metals (Mg++ or Mn++) for activity. • Highly thermodynamically favorable/reversible (DG°’ = –8.2 kcal/mol) – This reaction pulls the entire glycolytic pathway. – regulated by ATP, divalent metals, and other metabolites Glycolysis: Pyruvate Kinase (PK) Mechanism Phosphoryl transfer A=E315 R+ | Phosphoglycolate inhibitor http://clfs690.alivetek.org/CLFS690/glycolglucojmol/pyruvatekinase.htm Stereo views • Glu (E315) acts as an acid to protonate the enol • Arg (R116) acts to neutralize charges of phosphates during transfer • All kinases seem to have this Arg 7 Glycolysis: Summary Glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP • Used: – 1 glucose; 2 ATP; 2 NAD+, 2 ADP • Made: – 2 pyruvate • various different fates – 4 ATP • The net of 2 ATP is used for energy-requiring processes within the cell – 2 NADH • For glycolysis to continue, NADH must be re-oxidized • Glycolysis is heavily regulated. – ensure proper use of nutrients – ensure production of ATP only when needed – will discuss details after we do the opposite pathway (anabolism: gluconeogenesis ) Glycolysis: Isotope-labeling studies Experiment: If you give 14C-glucose to bacteria, with the label only on carbon #1, where would the label appear in GAP? * * 8 Glycolysis: Isotope-labeling studies * Experiment: If you give 14C-glucose to bacteria, with the label only on carbon #1, where would the label appear in pyruvate? – 1 2 Pyruvate 3 * Glycolysis: Chemical Logic H 2O 9 Glycolysis: Energetics Preparatory Phase -4 +0.4 -3.4 +5.7 Relative DG°’ DHAP & GAP Fru1,6P2 Fru6P Glc6P Glc Glycolysis: Energetics The Payoff Phase +1.8 -3.7 +1.1 +1.8 -8.2 Relative DG°’ (+1.8-5.5) PEP Pyruvate 2PG 1,3BPG à 3PG GAP Total for 1 Glc à 2 Pyruvate is –35 kcal/mol 10 Glycogenolysis & Glycolysis: Summary Debranching enzyme 2 Glycogen Phosphorylase PLP Pi 1 Phosphoglucomutase | OPO32– 3 Glucose 1-phosphate 2 Pi This slide has everything you need to commit to memory Mg2+ Mg2+ 13 steps 13 enzymes 12 structures 10 cofactors/co-substrates 11