Glycolysis Biochemistry by Reginald Garrett and Charles Grisham Chapter 18 Glycolysis Biochemistry by Reginald Garrett and Charles Grisham

18.1 – What Are the Essential Features of Glycolysis? The Embden-Meyerhof (Warburg) Pathway: Glycolysis Consists of two phases: First phase converts glucose to two Glyceraldehyde-3-P Energy investment phase Consumes 2 molecules of ATP Second phase produces two pyruvates Energy generation phase Produces 4 molecules of ATP Products are 2 pyruvate, 2 ATP and 2 NADH Essentially all cells carry out glycolysis Ten reactions - same in all cells - but rates differ

Figure 18.1 The glycolytic pathway.

Figure 18.2 Pyruvate produced in glycolysis can be utilized by cells in several ways. In animals, pyruvate is normally converted to acetyl-coenzyme A, which is then oxidized in the TCA cycle to produce CO2. When oxygen is limited, pyruvate can be converted to lactate. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.

18.2 – Why Are Coupled Reactions Important in Glycolysis? Coupled reactions convert some, but not all, of the metabolic energy of glucose into ATP The free energy change for the conversion of glucose to two lactates is -1863.6 Glucose  2 lactate + 2H+ DG0’ = -183.6 kJ/mol The production of two molecules of ATP in glycolysis is an energy-required process 2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol

Glycolysis couples these two reactions Glucose  2 lactate + 2H+ DG0’ = -183.6 kJ/mol 2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP + 2H+ + 2 H2O DG0’ = -122.6 kJ/mol More than enough free energy is available in the conversion of glucose into lactate to drive the synthesis of two molecules of ATP

18.3 – What Are the Chemical Principles and Features of the First Phase of Glycolysis?

Under cellular condition DG = DGo’ + RT ln ( [G-6-P][ADP] / [Glu][ATP] )

Phase 1 Phosphorylation (Hexokinase) Isomerization (Phosphoglucoisomerase) Phosphorylation (Phosphofructokinase) Cleavage (Aldolase) Isomerization (Triose phosphate isomerase)

Phosphorylation of glucose Reaction 1: Hexokinase Phosphorylation of glucose Hexokinase (or glucokinase in liver) This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Mg2+ is required Mg2+

The cellular advantages of phosphorylating glucose Phosphorylation keeps the substrate in the cell Keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell Makes it an important site for regulation Figure 18.4 Glucose-6-P cannot cross the plasma membrane.

Hexokinase 1st step in glycolysis; G large, negative Km for glucose is 0.1 mM; cell has 4 mM glucose, so hexokinase is normally active Hexokinase is regulated --allosterically inhibited by (product) glucose-6-P -- but is not the most important site of regulation of glycolysis—Why? (Figure 18.5) Can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose

Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.

Glucokinase The isozymes of hexokinase Hexokinase I in brain Hexokinase I (75%, 0.03mM) and II (25%, 0.3mM) in muscle Glucokinase in liver and pancreas Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose Is not product inhibited Is an inducible enzyme by insulin

Induced fit model (fig 13.24) Figure 18.6 The (a) open and (b) closed states of yeast hexokinase Figure 18.7 (a) Mammalian hexokinase I

Reaction 2: Phosphoglucoisomerase Glucose-6-P (aldose) to Fructose-6-P (ketose) Why does this reaction occur next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH isomerization activates C-3 for cleavage in aldolase reaction Phosphoglucose isomerase or glucose phosphate isomerase Ene-diol intermediate in this reaction

Phosphoglucoisomerase, with fructose-6-P (blue) bound.

Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)

Reaction 3: Phosphofructokinase PFK is the committed step in glycolysis The second priming reaction of glycolysis Committed step and large, negative DG -- means PFK is highly regulated Fructose-6-P + Pi → Fructose-1,6-bisP DGo’= 16.3 kJ/mol

Regulation of Phosphofructokinase ATP also is a allosteric inhibitor Has two distinct binding sites for ATP (A high-affinity substrate site and a low-affinity regulatory site) AMP reverses the inhibition due to ATP Raise dramatically when ATP decrease Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high

Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid.

Figure 18.10 Fructose-2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate concentration.

Figure 18.11 Fructose-2,6-bisphosphate decreases the inhibition of phosphofructokinase due to ATP.

Reaction 4: Fructose Bisphosphate Aldolase C6 is cleaved to 2 C3s (DHAP, Gly-3-P) Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates Dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P)

The aldolase reaction is unfavorable as written at standard state The aldolase reaction is unfavorable as written at standard state. The cellular ΔG, however, is close to zero. The aldolase reaction in glycolysis is merely the reverse of the aldol condensation well known to organic chemists.

Animal aldolases are Class I aldolases Class I aldolases form covalent Schiff base intermediate between substrate and active site lysine

Class II aldolase are produced mainly in bacteria and fungi

Reaction 5: Triose Phosphate Isomerase Only G-3-P goes directly into the second phase, DHAP must be converted to G-3-P Triose phosphate isomerase An ene-diol mechanism Active site Glu acts as general base is a near-perfect enzyme (Table 13.5)

Figure 18. 13 A reaction mechanism for triose phosphate isomerase Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu165.

18.4 – What Are the Chemical Principles and Features of the Second Phase of Glycolysis? Metabolic energy produces 4 ATP Net ATP yield for glycolysis is two ATP Second phase involves two very high energy phosphate intermediates 1,3 BPG Phosphoenolpyruvate Substrate-level phosphorylation

Phase 2 Oxidation and Phosphorylation (Glyceraldehyde-3-P Dehydrogenase) Substrate-level Phosphorylation (phosphoglycerate kinase) Isomerization (Phosphoglycerate isomerase) Dehydration (Enolase) Substrate-level Phosphorylation (pyruvate kinase)

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase G-3-P is oxidized to 1,3-BPG Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH Oxidation (aldehyde to carboxylic acid) and phosphorylation

G3PDH (or GAPDH) Mechanism involves covalent catalysis and a nicotinamide coenzyme

Reaction 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate This is referred to as "substrate-level phosphorylation" Coupled reactions; 6th and 7th reactions Glyceraldehyde-3-P + ADP + Pi + NAD+ → 3-phosphoglycerate + ATP + NADH + H+ DGo’= -12.6 kJ/mol

ATP is synthesized by three major routes: Substrate-level phosphorylation (Glycolysis, Citric acid cycle) Oxidative phosphorylation (Driven by electron transport) Photophosphorylation (Photosynthesis)

2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction Bisphosphoglycerate mutase Erythrocytes contain 4-5 mM 2,3-BPG Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate.

Reaction 8: Phosphoglycerate Mutase Repositions the phosphate Mutase: catalyzes migration of a functional group within a substrate

Phosphoenzyme intermediates A bit of 2,3-BPG is required as a cofactor The catalytic His183 at the active site of E. coli phosphoglycerate mutase

Reaction 9: Enolase The formation of PEP from 2-PG Dehydration Make a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis

Reaction 10: Pyruvate Kinase The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP. Substrate-level phosphorylation Another key control point for glycolysis Enol-keto tautomer .

Figure 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.

Large, negative G -- regulation Allosteric regulation Activated by AMP, F-1,6-bisP Inhibited by ATP ,acetyl-CoA, and alanine Liver pyruvate kinase is regulated by covalent modification Responsive to hormonally-regulated phosphorylation in the liver (glucagon) The phosphorylated form of the enzyme is more strongly inhibited by ATP and alanine. Has a higher Km for PEP

18.5 – What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? Aerobic or anaerobic? NADH must be recycled to NAD+ If O2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation (chapter 20) In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction.

Pyruvate also has two possible fates: aerobic: into citric acid cycle (chapter 19) where it is oxidized to CO2 with the production of additional NADH (and FADH2) anaerobic: (fermentation) In yeast: reduced to ethanol Pyruvate decarboxylase (TPP) Alcohol dehydrogenase (Reoxidized NADH to NAD+) In animals: reduced to lactate Lactate dehydrogenase (Reoxidized NADH to NAD+)

18.6 – How Do Cells Regulate Glycolysis? The elegant evidence of regulation (See Figure 18.22) Standard state G values are variously positive and negative G in cells is revealing: Most values near zero (reactions 2 and 4-9) 3 of 10 Reactions have large, negative G Large negative G Reactions are sites of regulation Hexokinase Phosphofructokinase Pyruvate kinase

Overview of the regulation of glycolysis.

18.7 – Are Substrates Other Than Glucose Used in Glycolysis? Sugars other than glucose can be glycolytic substrates Fructose and mannose are routed into glycolysis by fairly conventional means.

Fructose In liver In kidney and muscle Fructokinase Fructose + ATP  fructose-1-phosphate + ADP + H+ Fructose-1-phosphate aldolase fructose-1-phosphate  glyceraldehyde + DHAP Triose kinase glyceraldehyde  glyceraldehyde-3-phosphate In kidney and muscle Hexokinase Fructose + ATP  fructose-6-phosphate + ADP + H+

Mannose Hexokinase mannose + ATP  mannose-6-phosphate + ADP + H+ Phosphomannoisomerase mannose-6-phosphate  fructose-6-phosphate Galactose is more interesting - the Leloir pathway "converts" galactose to glucose Galactokinase Galactose + ATP  galactose-1-phosphate + ADP + H+ Galactose-1-phosphate uridylyltransferase Phosphoglucomutase UDP-galactose-4-epimerase

Figure 18.24 Galactose metabolism via the Leloir pathway.

Figure 18.25 The galactose-1-phosphate uridylyltransferase reaction involves a “ping-pong” kinetic mechanism.

Galactosemia Defects in galactose-1-P uridylyltransferase Galactose accumulate causes cataracts and permanent neurological disorders In adults, UDP-glucose pyrophosphorylase also works with galactose-1-P, reducing galactose toxicity

Lactose Intolerance The absence of the enzyme lactase (b-galactosidase) Diarrhea and discomfort

Glycerol can also enter glycolysis Glycerol is produced by the decomposition of triacylglycerols (chapter 23) Converted to glycerol-3-phosphate by the action of glycerol kinase Then oxidized to DHAP by the action of glycerol phosphate dehydrogenase NAD+ as the required coenzyme

18.8 How Do Cells Respond to Hypoxic Stress? Glycolysis is an anaerobic pathway—it does not require oxygen The TCA (tricarboxylic acid) cycle is aerobic. When oxygen is abundant, cells prefer to combine these pathways in aerobic metabolism When oxygen is limiting, cells adapt to carry out more glycolysis

Hypoxia (oxygen limitation) causes changes in gene expression that increases Angiogenesis (the growth of new blood vessels) Synthesis of red blood cells Levels of some glycolytic enzymes (a high rate of glycolysis) Hypoxic stress A trigger for this is a DNA binding protein called hypoxia inducible factor (HIF) HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1)

Hypoxia inducible factor (HIF) A heterodimer consists of two subunits: A constitutive, stable nuclear b subunit HIF-1β An inducible, unstable hypoxia-responsive HIF-α subunit Bind to the hypoxia responsive element (HRE) of hypoxia-inducible genes—Activating transcription of these genes HIF-α regulation is a multistep process Gene splicing Acetylation (Inhibited) Hydroxylation (Inhibited) Phosphorylation (activated)

Under normal oxygen levels, HIF-a are synthesized but quickly degraded When oxygen is plentiful, HIF-1a is hydroxylated by the prolyl hydroxylases (PHDs) These hydroxylation ensure its binding to ubiquitin E3 ligase, which leads to rapid proteolysis HIF-1a binding to the ubiquitin E3 ligase is also promoted by acetylation by the ARD1 acetyltransferase FIH-1 (hydroxylase factor-inhibiting HIF-a) hydroxylates HIF-a at Asn803 PHDs and FIH-1 both are oxygen-dependent

Figure 18.28