Glycogenolysis Glycolysis Fermentation Enolase

Glycolysis: Enolase – OH •Rationale: P – Dehydrates C3 to remove alcohol; more like pyruvate – Double-bonded C2-C3 is part of an en-ol except that the C2-alcohol is in ester linkage with a phosphate; without the alcohol hydrogen, it cannot tautomerize •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 1 Glycolysis: Enolase Mechanism Dehydration fast Base catalysis slow Acid catalysis – OH P P 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++) and K+ 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 2 Glycolysis: Pyruvate Kinase (PK) Mechanism Recall Phosphoryl transfer Mn2+ A=E315 + R+ K | =R116 P Phosphoglycolate inhibitor ⊝ 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 Glycolysis: Pyruvate Kinase (PK) Mechanism Stereo views 3 Glycolysis: Summary Glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O • Used: – 1 glucose; 2 ATP; 2 NAD+, 2 ADP, 2 Pi • 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? * * 4 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 5 Glycolysis: Energetics Preparatory Phase -4 +0.4 -3.4 +5.7 +1.8 Relative DG°’ GAP DHAP & GAP Fru1,6P2 Fru6P Glc6P Glc Glycolysis: Energetics The Payoff Phase x2 -3.7 +1.1 +1.8 -8.2 Relative DG°’ PEP Pyruvate 2PG 1,3BPG à 3PG GAP Total for 1 Glc à 2 Pyruvate is –18 kcal/mol 6 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+, K+ 13 steps 13 enzymes 12 structures 10 cofactors/co-substrates Catabolism of Other Sugars •Ingestion yields free glucose from glycogen and starch by a-amylase, maltase, and isomaltase •In the cell, glucose molecules are cleaved from glycogen and starch by glycogen phosphorylase. – yielding glucose 1-phosphate (and a little free Glc) – uses inorganic phosphate for lysis (phosphoro-lysis) •Disaccharides are hydrolyzed. – – – – lactose: glucose and galactose sucrose: glucose and fructose trehalose: glucose Monosaccharides fructose, galactose, and mannose enter glycolysis at different points. 7 Catabolism of Other Sugars Glc(a1àa1)Glc Gal(b1à4)Glc Glc(a1àb2)Fru Oligosaccharide Biosynthesis hexokinase MUSCLE LIVER UDP-mannose PGI hexokinase hexokinase a -Mannose 1-phosphate p ho sp ho m a n n o se m u ta se PFK-1 triose phosphate isom erase Aldolase/TIM Pyruvate Catabolism of Other Sugars ATP ADP Galactokinase Major cause of Galactosemia • Use of nucleotide sugars discovered by Argentinian biochemist Luis Leloir in 1940 for which he received the Nobel Prize. • Defects in the human genes for these enzymes causes what are called inborn errors in metabolism. • The cause of one of these due to galactose ingestion was one of the first discovered in 1956 by Herman Kalckar. • Galacotsemia causes enlarged liver, cirrhosis, kidney failure, cataracts, hypoglycemia, lethargy, and brain damage. Without treatment, mortality is about 75%. X • Defects in transferase are the underlying cause, with incidence of about 1:60,000 births. 8 Clinical Correlations Glycogen Storage Diseases: Inborn errors in metabolism Pompe is from a lack of maltase (a-1-4 glucosidase) in liver lysosomes, where glycogen will accumulate. Leads to death in first months after birth. Cori is from lack of debranching enzyme. Glycogen accumulates and liver enlargens (hepatomegaly). Also have fasting hypoglycemia. McArdle is from the absence of muscle phosphorylase. Leads to difficulty in muscle use; cramps, fatigue. Less muscle glycolysis leads to lower levels of lactic acid in the blood. Muscles are damaged and patients become debilitated. Clinical Correlations GAPDH: Arsenic poisoning and superoxide Arsenate (HAsO42–)s can substitute for Pi in biochemical reactions. But, the esters formed from arsenate are unstable and readily hydrolyze. For the GAPDH reaction then the 1-arsenyl-2-phospho glycerate degrades to 3-phosphoglycerate without production of ATP. Therefore, glycolysis does not net any ATP production. Arsenite (AsO2–) is more toxic. It kills any enzyme that contains lipoic acid: Superoxide (•O2–) overproduction can come from over-exposure to high blood glucose, especially in retina, kidney, and peripheral neurons.* This “reactive-oxygen species” (ROS) activates repair enzymes that end up modifying Cys residues in enzymes; thus killing GAPDH. 9