Biochemistry Ch 22 396-412
Glycolysis
-principal pathways for generating ATP in cells and is present in all cell types and has ability
to generate ATP with and without O2
-glucosepyruvate generates ATP via substrate-level phosphorylation and NADH
-pyruvate can be oxidized to CO2 in TCA cycle and ATP made from electron
transport
-in anaerobic glycolysis, pyruvate and NADH are converted to lactate and ATP made
without O2
-glucose can be found in diet, internal glycogen stores, and blood
-glucose is major fuel for all tissues except intestinal mucosal cells, brain uses during fasting
-glycolysis is an anabolic pathway providing precursors for synthesis of amino acids and 5P
Reactions of Glycolysis – cleaves 1 mol glucose  2 mol pyrivate through two phases:
1. Preparative Phase – glucose phosphorylated 2x by ATP and cleaved into 2 triose
phosphates
2. ATP-generating Phase – glyceraldehyde-3-phosphate (G3P) is oxidized by NAD+ and
phosphorylated using inorganic phosphate, and ADP ATP
a. The remaining phosphate is rearranged to form another ATP
b. Since 2 mole of G3P (Trioses) are formed, 4 mol total ATP and 2 mol NADH
formed, yielding a net 2 ATP, 2 NADH, 2 pyruvate from 1 glucose
1. Conversion of Glucose to Glucose-6-Phosphate- Glucose receives a phosphate from
ATP to form G6P with the help of hexokinase or glucokinase in liver
-commits the glucose to metabolism because G6P cannot travel back through membrane
-G6P is a branch point in carb metabolism: glycolysis, pentose phosphate, glycogen
synthesis
-hexokinases phosphorylate glucose; various isozymes that have different kinetic
properties
-isozyme in pancreas (glucokinase) has higher Km than other hexokinases
2. Conversion of G6P to Triose Phosphates – G6P  fructose-6-P by phosphoglucose
isomerase, phosphorylated again, and cleaved into two 3-carbon fragments
-F6P converted to fructose-1,6-bisP by phosphofructokinase-1, and is considered
the first committed step of the pathway (requires ATP)
-fructose-1,6-bisphosphate cleaved by aldolase into two triose phosphates
-dihydroxyacetonephosphate (DHAP) is isomerized to glyceraldehyde-3-phosphate
(a triose phosphate)
-for every mole of glucose, 2 mol of G3P are produced to continue pathway
3. Oxidation and Substrate-level Phosphorylation – G3P oxidized and phosphorylated
so that subsequent intermediates can donate phosphate to ADP  ATP
a. G3P-dehydrogenase – oxidizes aldehyde in G3P and transfers electrons to
NAD+  NADH
-forms high energy phosphate bond, which is the start of substrate-level
phosph
b. Phosphoglycerate kinase transfers phosphate from ADP to ATP and forms a 3phosphoglycerate product.
c. 3-phosphoglycerate converted to 2-phosphoglycerate by
phosphoglyceromutase which is then converted to phosphoenol pyruvate (PEP)
by enolase and finally to pyruvate by pyruvate kinase and transfers phosphate to
ADP  ATP
SUMMARY OF GLYCOLYSIS
Glucose  G6P  F6P  F1,6bisP  G3P  1,3 BPG  3-PG  2-PG  PEP  Pyruvate
Enzymes
Hexokinase, phosphoglucose isomerase, PFK-1, aldolase, triose phosphate isomerase,
glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase,
phosphogluceromutase, enolase, pyruvate kinase
RED = ATP GENERATED BLUE = NADH GENERATED
GLUCOSE + 2NAD + 2Pi + 2ADP  2 PYRUVATE + 2 NADH + 4H + 2 ATP + 2 H2O
Oxidative Fates of Pyruvate and NADH – NADH must be reoxidized to NAD+ to continue
reaction. NADH can be reoxidized aerobically and anaerobically
-Aerobic NADH: shuttles NADH across mitochondrial membrane for electron
transport (mit. Membrane impermeable to NADH)
-Anaerobic NADH: NADH reoxidized by lactate dehydrogenase reducing pyruvate
to lactate
-fate of pyruvate depends on route of NADH oxidation; if NADH is used aerobically,
pyruvate can be used for TCA cycle by its oxidation to acetyl-CoA.
-NADH reoxidized to NAD+ in cytosol by reaction that transfers electrons to DHAP
in glycerol-3-P shuttle and oxaloacetate in malate-asparate shuttle. NAD formed in
cytosol returns to glycolysis and glycerol-3-P or malate carries reducing equivalent
across mitochondrial membrane (electrons, not NADH)
-if NADH is used anaerobically, pyruvate is reduced to lactate
Glycerol 3-Phosphate Shuttle – major shuttle in most tissues where NAD is regenerated
by glycerol-3P dehydrogenase which transfers electrons from NADH to DHAP to form
glycerol 3-P, whichdiffuses through outer mit. Membrane to the inner membrane where
electrons are donated to a FAD-glycerophophsate dehydrogenase, donating electrons to
coenzyme Q resulting in 1.5 ATP from OxPhos.
NADH(cyt) + H + FAD (mit)  NAD(cyt) + FADH2(mit)
Malate-Aspartate Shuttle – cytosolic NAD is regenerated by malate dehydrogenase which
transfers electrons from NADH to cytosolic oxaloacetate  malate
-malate is transferred across inner mit membrane and exchanges malate for alphaketoglutarate
-in matrix, malate is oxidized back to oxaloacetate by mitochondrial malate dehydrogenase
and NADH is generated (which can donate electrons to ETC)
NADH (cytosol) + NAD (matrix)  NAD (cytosol) + NADH (matrix)
Anaerobic Glycolysis – NADH produced from glycolysis cannot be oxidized aerobically and
thus is oxidized back to NAD+ by lactate dehydrogenase.
GLUCOSE + 2ADP + 2Pi  2 LACTATE + 2 ATP + 2H2O + 2H
-each mol of glucose generates 2 mol ATP, 2 mol NADH, and 2 pyruvate
-in anaerobic metabolism, 1 glucose forms 2 moles of lactate and only 2 mol ATP per
glucose
-NADH nor pyruvate are used for further energy
-oxidation of pyruvate in TCA generates 12.5mol ATP per mol pyruvate, 1.5 mol of
ATP generated (Glycerol-3-phosphate shuttle) or 2.5 mol ATP from malateaspartate shuttle from aerobic oxidation of cytosolic NADH, therefore, TCA produces
30-32 mol ATP per glucose
Acid Production in Anaerobic Glycolysis – results in acid production in the form of
hydrogen ions. Glycolysis forms pyruvic acid  lactic acid, which dissiates to form
carboxylate anion, lactate, and H+, which can reduce pH
Tissues Dependent on Anaerobic Glycolysis – RBCs, kidney medulla, eye, skeletal
muscles, all rely on anaerobic glycolysis for a portion of ATP requirements, and they have a
low ATP demand to begin with.
-some lactic acid generated in skin is secreted as sweat, acting as antibacterial agent
-tissues with mitrochondria show both aerobic and anaerobic glycolysis
Fate of Lactate – lactate is taken up by other tissues (liver, heart, skeletal muscle) and
oxidized back to pyruvate
-in the liver, pyruvate synthesizes glucose (gluconeogenesis), cycling of lactate and glucose
between liver and peripheral tissues is called the Cori Cycle
-in other tissues, lactate is oxidized to pyruvate and then to CO2 in TCA cycle
-LACTATE + NAD  PYRUVATE + NADH + H
-the heart uses lactate as fuel; similarly, skeletal muscles can use lactate during exercise
-in the brain, glial cells and astrocytes produce lactate which is used by neurons
-lactate dehydrogenase is tetramer composed of A-subunits, B subunits which combine in
random combinations
-M4 form (4 A subunits) is used in skeletal muscle, H4 in heart
Other Functions of Glycolysis – provides precursors for biosynthetic pathways, such as for
ribose-5-phosphate (for nucleotides), UDP-glucose, mannose, and sialic acid
-serin is synthesized from 3-phosphoglycerate, and alanine from pyruvate
-triacylglycerols, glycerol-3-P converted from DHAP
-liver is site of biosynthetic pathways in the body
-bisphosphoglycerate shunt is a side reaction of glycolytic pathway in which 1,3 BPG is
converted to 2,3BPG to serve as an allosteric inhibitor of O2 binding to heme
-2,3 BPG reenters glycolysisas 3-phosphoglycerate
Regulation of Glycolysis by the need for ATP – pathway is designed to regulate and
maintain ATP homeostasis in cells
-PFK-1 and PDH links glycolysis and TCA cycle are major regulatory sites that
respond to feedback indicators of ATP utilization
-supply of G6P is tissue dependent and is regulated by glycogenolysis and rate of
phosphorylation by hexokinase enzymes
Relationships among ATP, ADP, AMP Concentrations – AMP levels provide a better
indicator of rate of ATP utilization than ATP concentrations itself
2 ADP  AMP + ATP
-AMP activates glycolysis, glycogenolysis, fatty acid oxidation to maintain ATP homeostasis
Regulation of Hexokinases – hexokinases are normally inhibited by glucose-6-phosphate,
and if G6P does not enter glycolysis or other pathways, it accumulates and inhibits
hexokinase
-in liver, Km of glucokinase is high, so glycolysis can continue even if energy is high
Regulation of Phosphofructokinase-1 – PFK-1 is a rate-limiting step of glycolysis that
regulates rate of glucose-6-P entry into glycolysis
-PFK1 is an allosteric enzyme with 6 binding sites, two for substrates and 4 for
regulatory sites
-AMP and F-2,6-bisP are ACTIVATORS
-ATP, citrate are INHIBITORS
-PFK comes as isozymes called M (muscle), L (liver), and C (other tissues)
-AMP increases affinity of PFK-1 for fructose-6-P (shifts kinetic curve to the left)
-F-2,6-bisP opposes ATP inhibition and is not an intermediate in glycolysis by is synthesized
by an enzyme that phosphorylates F6P at 2-position (PFK-2)
-PFK2 is regulated through change in ratio of activity of two domains (phosphatase
and kinase)
-PFK2 can also be regulated through phosphorylation, which can increase kinase
activity and increase fructose 2,6-bisP levels to contribute to glycolysis activation
Allosteric Inhibition of PFK-1 at Citrate Site – function of citrate allosteric site on PFK1 is
to integrate glycolysis with other pathways
Regulation of Pyruvate Kinase – pyruvate kinase exists are R (RBC), L (Liver), M1/2
(muscle) isozymes have various allosteric sites
-liver pyruvate kinase can be inhibited through phosphorylation by cAMP kinase and
activated by other effectors (F-1,6-bisP)
Pyruvate Dehydrogenase Regulation and Glycolysis – PDH is regulated principally by
rate of ATP utilization through rapid phosphorylation to an inactive form
-glycolysis and TCA are regulated together in a cell in adequate oxygen. In low O2,
NADH/NAD ratio inhibits PDH, but AMP activates glycolysis
-portion of pyruvate is then reduced to lactate to allow glycolysis
Lactic Acidemia – in lactic acidosis, lactic acid accumulates in blood to levels that
significantly affects pH
-lactic acidosis results from greatly increased NADH/NAD ratio in tissues
-increased NADH concentrations prevens pyruvate oxidation in TCA cycle and directs
pyruvate to lactate
-to compensate for decreased ATP from OxPhos, PFK-1 is activated and glycolysis activated
-drinking alcohol increases NADH levels and can lead to lactic acidosis
-hypoxia increases lactate production
-Impaired PDH activated from inherited deficiency of E1 (subunit in complex), or from
severe thiamine deficiency, increases blood lactate levels
-Pyruvate carboxylase deficiency can lead to lactic acidosis