Download Lecture 12-14 (Parker) - Department of Chemistry ::: CALTECH

Glycolysis is the sequence of reactions that metabolize one molecule of
glucose into two molecules of pyruvate with the production of two
molecules of ATP
Under anaerobic conditions pyruvate can be processed either
to ethanol or lactate
Under aerobic conditions pyruvate can be completely
oxidized to CO2
In 1897 Hans and Eduard Buchner discovered that yeast extracts
could rapidly ferment sucrose into alcohol. Startling discovery in
its day because it was widely held that fermentation was a process
that occurs within cells.
This led to efforts to understand the mechanism of this
extracellular fermentation. Studies in muscle extracts also
revealed that lactic acid fermentation was very similar to alcohol
fermentation in yeast.
The complete glycolytic pathway was finally elucidated by 1940
through the pioneering contributions of Embden, Meyerhof,
Neuberg, Parnas, Warburg and G&C Cori.
Glycolysis is also known as the Embden-Meyerhof pathway.
Glucose is an important fuel for most organisms.
In mammals glucose is used by the brain under nonstarvation conditions and is the only fuel used by red
blood cells.
There are many different monosaccharides but glucose
predominates as the primary fuel.
Glucose is generated from dietary carbohydrates:
Pancreatic a-amylase cleaves the 1,4 bonds of starch the
products are maltose and maltotriose.
Maltase cleaves maltose into two glucose molecules and aglucosidase cleaves maltotriose into three glucose molecules.
Maltase and a-glucosidase are located on the surface of the
small intestine these monosaccharides are now transported into
the blood stream.
In eukaryotic cells glycolysis takes place in the cytoplasm in
two stages:
Glucose enters cells through specific transport proteins after
entry it is immediately phosphorylated by hexokinase and ATP
to form glucose 6-phosphate.
This traps the glucose into the cell because G6P cannot pass
through the cell membrane and destabilizes it.
Induced fit in hexokinase
Hexokinase places glucose into a cleft and then the cleft
closes trapping glucose and excluding H2O
Conversion of G-6P into F-6P requires two steps by
phosphoglucose isomerase:
1)  The glucose chain must be opened
2)  Isomerization to the open chain aldose to ketose F-6P
3)  Then a 4 carbon atom ring closure occurs leaving closed ring
F-6P
A second phosphorylation step occurs after isomerization
catalyzed by phosphofructokinase:
Essentially non-reversible
The 6-carbon F-1,6-BP is cleaved by aldolase into G3P and
DHAP
G3P is on a direct pathway of glycolysis whereas DHAP is not and needs
to be converted into G3P by an isomerase:
At equilibrium 96% of the triose phosphate is DHAP but utilization of
G3P by glycolysis forces the reaction to generate G3P
Structure of triose phosphate isomerase
Glu165 and His95 play key roles in the reaction
Glu removes the H+ from C1 and His donates a H+ to the O bound to C2
Glu165 and His95 play key roles in the reaction
Glu removes the H+ from C1 and His donates a H+ to the O
bound to C2
TPI traps the enediol intermediate with the adjacent loop of the enzyme
preventing loss of the intermediate which would lead to the production of a
toxic methyl glyoxal molecule
STAGE 1
STAGE 2
Oxidation of the aldehyde to and acid
powers the formation of the high
phosphoryl-transfer potential 1,3-BPG
Glyceraldehyde 3-P dehydrogenase
synthesizes 1,3-BPG in two steps:
Catalytic mechanism of glyceraldehyde 3-phosphate
dehydrogenase
Cys reacts with the
aldehyde group of
the substrate
Oxidation takes
place with the
transfer of H+ to
NAD forming a
thioester
intermediate
NADH is replaced by NAD+
Orthophosphate attacks the thioester
forming 1,3-BPG
ATP is formed by phosphoryl transfer from 1,3-BPG to ADP
catalyzed by PGK
Additional ATP is generated with the formation of pyruvate
The phosphoryl group present in the enol form generated by the
action of enolase is unstable; once transferred to ADP by pyruvate
kinase it rapidly undergoes conversion to the ketone pyruvate.
The driving force for this reaction is large because of this
conversion.
Glucose+2Pi+2ADP+2NAD == 2pyruvate+2ATP+2NADH+2H+
NAD+ is regenerated from the metabolism of pyruvate
Ethanol is formed from pyruvate in yeast and other
microorganisms
Lactate is formed in a variery of microorganisms and also in cells
of higher organisms when 02 is limiting
Much more energy is extracted by conversion of pyruvate to acetyl
CoA via the citric acid cycle
Pyruvate to ethanol regenerates NAD+
The regeneration of NAD+ by the alcohol and lactate
fermentation keeps a redox balance
The regeneration of NAD+ by the alcohol and lactate
fermentation keeps a redox balance
The three dehydrogenases that we have considered are very
different structurally but all three possess a conserved NAD+
binding domain
4 enzymatic
steps
1 enzymatic
step
Fructose in the liver enters the glycolytic pathway via
the fructose 1-phosphate pathway
THIS
PATHWAY
BYPASSES
PFK NO ATP
FEEDBACK
TPI
The fructose 1 phosphate pathway in the liver by passes ATP
feed back allowing high levels of AcetylCoA to accumulate
leading to the production of fatty acids and a fatty liver this
occurs if consumption of high fructose corn syrup is high
Galactose is converted into glucose 6-phosphate in four steps
Entry of galactose into glycolysis utilizes a previously
modified galactose molecule
PGM
Lactose intolerance is due to a low level of lactase
A deficency of galactose 1-phosphate uridyl transferase can
lead to toxic levels of galactose; in the eye lens galactose is
converted into galactitol causing the production of a cataract
The Glycolytic pathway is tightly controlled
The rate of conversion of glucose to pyruvate is tightly
regulated.
In glycolysis the reactions catalyzed by hexokinase,
phosphofructokinase and pyruvate kinase are essentially
irreversible making them targets for regulation.
These enzymes become more or less active in response to the
binding of allosteric effectors (milliseconds) or covalent
modifications (seconds).
In addition the levels of these enzymes are regulated by the
transcription of their genes (minutes).
Phosphofructokinase is regulated by the ratio of
ATP/AMP
A tetramer
of 4 identical
subunits
At high levels of ATP it will bind to an allosteric site reducing
the enzyme’s affinity for fructose 6-PO
AMP reverses the inhibitory action of ATP
Hexokinase is inhibited by high levels of its product
glucose 6-phosphate
The regulation of glycolysis in the liver is more complex than
in muscle tissue
The liver has more diverse biochemical functions than muscle.
The liver maintains blood-glucose levels and stores glucose as
glycogen when glucose levels are plentiful.
Phosphofructokinase regulation with respect to ATP is similar to
muscle, however low pH is not a metabolic signal.
In the liver phosphofructokinase is inhibited by citrate, an early
intermediate in the citric acid cycle.
The regulation of glycolysis in the liver is more complex than
in muscle tissue
A high level of citrate indicates that biosynthetic intermediates
are abundant so there is no need to degrade additional glucose
for this purpose.
Citrate inhibits phosphofructokinase by enhancing the inhibitory
effect of ATP.
Glycolysis in the liver responds to changes in blood glucose
through the signaling molecule; fructose 2,6-bisphosphate.
F-2,6-BP is a potent activator of phosphofructokinase.
In the liver the concentration of fructose 6-phosphate rises when
blood glucose is high, the abundance of fructose 6-phosphate
accelerates the synthesis of F-2,6-BP.
F-2,6-BP increases the affinity of phosphofructokinase and
diminishes the inhibitory effect of ATP.
Thus glycolysis is accelerated when glucose is abundant, a process
known as feed-forward stimulation.
Feed-forward stimulation in response to high blood glucose
levels by the liver
F-2,6-BP stimulates phosphofructokinase even at low substrate
concentrations (A). ATP inhibitory effects are attenuated in the presence
of F-2,6-BP (B).
The liver isoforms of pyruvate kinase can be modified by phosphorylation
which reduce its activity and limit the livers utilization of glucose saving it for
muscle and brain to consume.
GLUT’s consist of a single chain of 500 amino acid isoforms:
Glucose can be synthesized from non-carbohydrate
precursors
The gluconeogenesis pathway converts pyruvate into glucose.
The major non-carbohydrate precursors are lactate, amino acids
and glycerol.
Lactate formed in muscle tissue and can be readily converted into
pyruvate by the enzyme lactate dehydrogenase.
Amino acids are derived from proteins in the diet.
Glycerol is converted into dihydroxyacetone phosphate then
enters the pathway via conversion to glyceraldehyde 3-phosphate
Pathway of
Glycolysis
Pathway of
Gluconeogenesis
Conversion of glycerol to dihydroxyacetone phosphate
Pyruvate is converted into oxaloacetate by a mitochondrial
enzyme: pyruvate carboxylase
Domain structure of pyruvate carboxylase
Biotin-binding domain of pyruvate carboxylase; biotin is
covalently attached through an e-nitrogen atom of lysine
Biotin contains an activated CO2 molecule
pyruvate
carboxylase
Malate
dehydrogenase
Oxaloacetate is converted into phosphoenolpyruvate by an ER
bound enzyme: phosphoenolpyruvate carboxykinase
Glucose 6-phosphate is converted to glucose in the ER by glucose
6-phosphatase
This process occurs primarily in the liver:
The stoichiometry of gluconeogenesis:
2pyruvate+4ATP+2GTP+2NADH+6H2O=
glucose+4ADP+2GDP+6Pi+2NAD+2H+
DG=-11 kcal/mole
Energy charge determines whether glycolysis of gluconeogenesis
will be most active
The most important regulatory site is the interconversion of fructose
6-phosphate and fructose 1,6-bisphosphate
When energy is needed the concentration of AMP will be relatively
high. AMP stimulates phosphofructokinase and inhibits fructose
1,6-bisphosphatase, glycolysis is favored.
Conversely high levels of ATP and citrate indicate there are
abundant biosynthetic intermediates. ATP and citrate inhibit
phosphofructokinase and activate fructose 1,6-bisphosphatase,
gluconeogenesis is favored.
Glycolysis and gluconeogenesis are also reciprocally regulated at
the interconversion of phosphoenolpyruvate and pyruvate in the
liver
The glycolytic enzyme pyruvate kinase is inhibited by allosteric
effectors ATP and alanine, signaling energy charge is high and
building blocks are abundant.
Conversely pyruvate carboxylase, which catalyzes the first step of
gluconeogenesis from pyruvate is inhibited by ADP. Similarly
ADP inhibits phosphoenolpyruvate carboxykinase.
Gluconeogenesis is favored when a cell has ample ATP and
biosynthetic precursors.
The balance between glycolysis and gluconeogenesis in the
liver is sensitive to blood-glucose concentrations
The signaling molecule fructose 2,6-bisphosphate strongly
stimulates phosphofructokinase(PFK) and inhibits fructose 1,6bisphosphatase
When blood-glucose is low fructose 2,6-bisphosphate looses a
phosphate to form fructose 6-phosphate which can not bind to
PFK.
The level of fructose 2,6-bisphosphate is critical in determining
whether glycolysis or gluconeogenesis will occur. Two enzymes
regulate the concentration of this molecule: one phosphorylates
fructose 6-phosphate and the other dephosphorylates fructose 2,6bisphosphate.
Domain structure of the bifunctional enzyme phosphofructokinase 2
The activities of PFK2 and FBP2 are reciprocally controlled by
the phosphorylation of a single serine residue
At low glucose levels a rise in the hormone glucagon triggers a
cAMP signaling cascade leading to the phosphorylation of the
bifunctional enzyme by protein kinase A. This modification
activates FBPase2 and inactivates PFK2 leading to a drop in the
levels of F-2,6-BP resulting in gluconeogenesis.
When blood glucose levels are high, insulin is secreted by the
pancreas initiating a signaling pathway that activates a protein
phosphatase removing the phosphoryl group from the
bifunctional enzyme thus PFK2 is activated and FBP2 inhibited
and glycolysis begins as the levels of F-2,6-BP rise.
Promoter structure of the phosphoenolpyruvate carboxykinase
gene
IRE insulin response element (repression)
GRE glucocorticoid response element
TRE thyroid hormone response element
CREI and CREII cAMP response elements (activation)
The Cori cycle
Lactate is converted to pyruvate by lactate dehydrogenase
Isozymic forms of lactate dehydrogenase in different tissues
catalyze the inter-converson of pyruvate to lactate.
The two primary isoforms are H (predominates in the heart) and
M (predominates in skeletal muscle and the liver).
These subunits associate to form 5 types of tetramers the H4 has
higher affinities for substrates than the M4.
H4 oxidizes lactate to pyruvate so the heart is always aerobic.
M4 is optimized to convert pyruvate into lactate to allow
glycolysis to proceed under anaerobic conditions.