Metabolism Flashcards
Glycolysis location
Cytoplasm
Glycolysis equation
Glucose + 2ADP +2Pi + 2NAD+ —> 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
What is substrate level phosphorylation
Direct transfer of a phosphate group onto ADP
From a molecule with higher phosphoryl transfer potential
So free energy released is higher than free energy of hydrolysis of atp
Importance of glycolysis
Energy production in anaerobic conditions
3 control points in glycolysis
Hexokinase (glucose to G6P)
Phosphofructokinase (PFK) (makes fructose 1.6 bp)
Pyruvate kinase (PEP to pyruvate)
Isoenzymes of PFK (glycolysis)
2 different forms of an enzyme that catalyse the same reaction
Can be regulated differently
Different isoenzymes in muscle and liver
In muscle decreasing pH decreases enzyme activity
In liver, citrate is an Allosteric inhibitor and fructose 2,6 BP is an Allosteric activator
Allosteric regulation of PFK (glycolysis)
Regulated by need for atp in muscles
ATP inhibits
AMP activates
ATP decreases affinity for fructose-6-P
Regulation of pyruvate kinase in liver (glycolysis)
Regulated by phosphorylation
Usually stimulated by hormones
Phosphorylated form less active
Phosphorykated by Cyclic AMP dependent kinase (PKA)
Activated by phosphoprotein phosphatase
PKA stimulated by glucagon
Can phosphorylate serine residue
Function of tca cycle
Final common pathway for oxidation of all fuel molecules
Complete oxidation of acetyl-coA
Produces reduced cofactors that carry e- to ETC
Directly generates atp by substrate level phosphorylation
Net reaction of TCA cycle
Acetyl-coA + 3NAD+ + FAD +ADP (GDP) + Pi + 2H2O —> 2CO2 + 3NADH + FADH2 + ATP (GTP) + CoA
How many ATP from NADH
2.5 ATP
How many ATP from FADH2
1.5 ATP
How is ATP generated in oxidative phosphorylation
NADH and FADH2 reoxidised in ETC
E- passed to ETC components then finally to O2
Process coupled to ATP production
Bio synthetic reactions of tca cycle
Citrate —> FAs/ sterols
Alpha-keto glutarate —> aas —> purines
Succinyl-CoA —> porphyrins —> haem
OAA —> glucose / aas/ purines/pyrimidines
Reactions happen in cytoplasm so must be carriers in IMM for intermediates to move out
Replenishing OAA for TCA
Carboxylation of pyruvate by pyruvate carboxylase
Has biotin attached
Bicarbonate + pyruvate —> OAA
Control of TCA cycle
Allosterically regulated
Responds to energy charge ( ATP/ADP ratio)
Controlled enzymes:
Pyruvate dehydrogenase
Isocitrate dehydrogenase
Alpha keto glutarate dehydrogenase
Allosteric control of pyruvate dehydrogenase ( TCA)
ATP inhibits, NADH inhibits, acetyl-CoA inhibits
ADP activates, pyruvate activates
Allosteric control of isocitrate dehydrogenase (TCA)
ATP inhibits, NADH inhibits
ADP activates
Allosteric control of alpha keto glutarate dehydrogenase (TCA)
NADH inhibits, ATP inhibits
Control of pyruvate dehydrogenase by phosphorylation (TCA)
Inactivated by phosphorylation on serine residue in E1 subunit
Phosphorylated by pyruvate dehydrogenase kinase
Dephosphorylated by pyruvate dehydrogenase phosphatase
Kinase and phosphatase controlled by Allostery and hormones
Kinase:
Activated by NADH, ATP Acetyl-CoA
Inhibited by NAD+, CoA, ADP, Ca2+ (muscle)
Phosphatase:
Activated by Ca2+ in muscles
Activated by insulin in liver
Glycogen synthesis
UDP glucose synthesised from Glucose-1-P and UTP
Made from UDP-glucose
Initiation by glycogenin
Tyr 194 nucleophilic attack onto UDP sugar (glucosyltransferase activity)
Chain extending activity adds a glucose at non-reducing end (where CH2 is)
Repeats x6
Glycogen synthase catalyses formation of alpha-1,4 glycosidic linkage
UDP displaced by OH group in C4
Branching by Glycosyl 4-6 transferase
Breaks 1-4 bond and moves chain to create 1-6 linkage
Transfers terminal 6/7 residues from non-reducing end
Need chain of at least 11 residues
Glycogen breakdown
Glycogen phosphorylase adds a phosphate across the 1-4 bond and removes a residue from non-reducing end by phospholytic cleavage
Releases glucose-1-P
Pyridoxal phosphate co factor involved in acid base catalysis
Can take off residues until within 4 residues of a branch
Transferase activity of debranching enzymes moves 3 residues to non reducing end
1-6 glucosidase activity of debranching enzyme hydrolyses 1-6 bond and releases free glucose
Glucose 1-p converted to glucose-6-p via glucose-1,6-bp by phosphoglucomutase
Glucose-6-p used in muscle for glycolysis for a higher net atp or liver hydrolyses back to free glucose
Regulation of glycogen phosphorylase
2 forms, phosphorylase a and phosphorylase b
A is double phosphorylated and favours R state
B is less active and favours T state
Interconverted by phosphorylase b kinase and phosphoprotein phosphatase 1
Phosphorylase b kinase activated by glucagon and adrenaline
Phosphoprotein phosphatase 1 activated by insulin
Also Allosteric regulation
In muscle:
Phosphorylase b inhibited by glucose-6-p and ATP, only active if AMP high
In liver:
Phosphorylase a inhibited by glucose, enzyme acts as glucose sensor
Regulation of glycogen synthase
2 subunits
Each one is triple phosphorylated
Phosphorylated form is glycogen synthase b, less active
Dephosphorylated form is glycogen synthase a, more active
Interconverted by phosphoprotein phosphatase (PP1) and glycogen synthase kinase (GSK1)
Glucagon and adrenaline activate kinase
Insulin activates PP1 in liver and muscle
Insulin can inactivate GSK3
Allosteric regulation:
G6P is an Allosteric regulator of glycogen synthase b
Signalling pathway for glucagon and adrenaline
GCPR receptor
Conformational change as GDP exchanged for GTP
Alpha subunit disssociates
Binds adenylate cyclase
ATP—> cAMP
Activates PKA
Phosphorylates phosphorylase b kinase and glycogen synthase a
Insulin signalling pathway
Tyrosine kinase receptor
Heterodimer
When insulin binds, 2 intracellular tyrosine kinases phosphorylate each other
Activates protein kinase
Inactivates SK 3
Activates PP1
Gluconeogenesis net reaction
2 pyruvate + 4 ATP + 2GTP + 2NADH + 2H+ + 4H2O —> glucose + 4ADP + 4Pi + 2GDP + 2Pi + 2 NAD+
Energetically very costly but essential
6 ATP for each glucose made
Precursors of glucose
Lactate —> pyruvate
Some amino acids e.g. alanine —> pyruvate
Glycerol from triacylglycerols —> G3P
Cori Cycle
In liver:
2 lactate —> 2 pyruvate —> glucose
Lactate oxidised to pyruvate
Travels in blood
In muscle:
Glucose —> 2 pyruvate —> 2 lactate
Pyruvate reduced to lactate in anaerobic conditions
Travels in blood
Cycle shifts metabolic burden of glucose synthesis from RBC and muscles to liver
Why do we need reciprocal regulation of glycolysis and gluconeogenesis
If both reactions happened at the same time you would get a futile cycle
Hydrolysis of atp with no useful metabolic reaction occurring is wasteful
Regulation of fructose-6-p to fructose-1,6-bp
F6p-f16bp by phosphofructose kinase
Activated by F2,6BP, AMP
Inhibited by ATP, citrate, H+
F1,6BP to F6P by fructose 1,6 bisphosphatase
Activated by citrate
Inhibited by F2,6BP and AMP
Synthesis of fructose 2,6 BP from fructose 6 p is controlled by hormones
Regulation of PEP —> pyruvate interconversion
PEP—-> pyruvate by pyruvate kinase
Activated by F 1,6 BP
Inhibited by ATP and alanine
Pyruvate —> OAA by pyruvate carboxylase
Activated by acetyl-CoA
Inhibited by ADP
OAA—> PEP by PEP carboxykinase
Inhibited by ADP
Regulation of PFK2 and FBPase 2 by hormones
Catalyse interconversion of fructose - 6 -p and fructose -2,6 bp
Both in one bifunctional enzyme
Regulated by phosphorylation
Phosphorylated form has inactive PFK 2 and active FBPase 2
Phosphorylated by cAMP dependent kinase
Glucagon stimulates production of cAMP so activates the kinase
Phosphatase activated by insulin
Processes of lipid breakdown
Mobilisation of Fas from TGs in adipose tissue
Transport to tissues by serum albumin
Activation of FAs by CoA
Transport into matrix - requires conjugation to carnitine
Beta oxidation of FAs produces acetyl coA
Acetyl coA oxidised in TCA cycle
Control of release of FAs from adipose tissue
Controlled by hormones
Glucagon and adrenaline bind GCPR receptors
Cascade
CAMP activates PKA which phosphorylates TRIACYLGLYCEROL LIPASE
Releases a free fatty acid from TG
Diacylglycerol and monoacylglyceol lipase act on diacylglycerol to release free FAs and glycerol
Free FAs diffuse through membrane into blood
Very hydrophobic so once in blood they bind to serum albumin which has many hydrophobic binding sites
Conjugation of FAs to carnitine and transport into matrix
Acyl-coA + carnitine undergo transesterification reaction to swap thiol for carnitine group
Acyl-coA can go through pore in OMM
Carnitine acyltransferase I in OMM catalyses formation of Acyl-carnitine in IMS
Carnitine in IMS
Acyl-carnitine travels into matrix through translocase in IMM
Carnitine acyltransferase II in IMM catalyses conversion of Acyl-carnitine to Acyl-coA in matrix
Carnitine travels back through translocase to IMS
Beta oxidation of FAs
Series of 4 reactions
Releases Acetyl-coA
Oxidation by FAD of beta carbon to create double bond
Hydration across double bond
Oxidation with NAD+ so hydroxyl —> keto
Thiolytic cleavage as CoA reacts with 2C
For odd number chains:
Carboxylation produces D-methylmalonylcoA
Isomerisation to L isomer
Mutase converts to succinyl CoA that enters TCA
Net reaction for beta oxidation of palmitate (C16)
Palmityl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O —> 8 acetyl-coA + 7FADH2 + 7NADH + 7H+
Hormonal control of TG lipase (FA degradation)
Activated by glucagon or adrenaline
Inhibited by insulin
Control of Transport of FAs into mitochondria
Carnitine acyltransferase I inhibited by malonyl coA
High malonyl coA corresponds to high acetyl-coA so dont need more
FAs can’t move into matrix
FA synthesis committed step
Acetyl-coA + bicarbonate + ATP —> malonyl coA
By acetyl-coA carboxylase
Elongation phase of FA synthesis
Malonyl coA/ acetyl-coA transferases swap coA for same group attached to ACP
Condensation - malonyl-CoA is activated donor of 2C units, release of CO2 drives reactions, forms 4CFA-ACP
Reduction reaction to reduce ketone to hydroxyl using NADPH
Dehydration to create double bond
Reduction to reduce C=C using NADPH
Repeated cycle of 4 reactions with malonyl-acp each time 2C added
Can repeat up to 7 times
Hydrolysis of thio-ester bond to release FA from ACP
Overall reaction for palmitate synthesis
Acetyl-ACP + 7 malonyl-ACP + 14 NADPH + 14 H+ —> palmitate + 7CO2 + 14 NADP+ 8ACP + 6H2O
To make malonyl-coA:
7acetyl-coA + 7CO2 + 7ATP —> 7malonyl-CoA + 7ADP + 7Pi
Overall:
8 acetyl-coA + 7ATP + 14 NADPH + 14H+ —> palmitate + 14 NADP+ + 8CoA + 7ADP + 7Pi + 6H2O
Energetically costly
Transport of acetyl-coA out of mitochondria
No carrier in IMM so citrate acts as a carrier of acetyl coA
In matrix:
Malate oxidised to OAA
OAA + acetyl-coA —> citrate
Citrate moves to cytoplasm when atp high
Citrate —> OAA + acetyl coA using 1 ATP
OAA reduced to malate
Malate —> pyruvate
Pyruvate moves to matrix
Control of acetyl-coA carboxylase (FA synthesis) by phosphorylation
Phosphorylated form is inactive
Phosphatase activated by insulin
Kinase is AMP dependent, activated by AMP and inhibited by ATP
Also activated by glucagon and adrenaline in cascade
Allosteric control of acetyl-coA carboxylase (FA synthesis)
Phosphorylated form is allosterically activated by citrate to make it partially active
Citrate conc high when TCA cycle is slow
Effect of citrate inhibited by palmitoyl-coA
Glucogenic amino acids
Can make glucose in gluconeogenesis and be oxidised in TCA cycle
Gly, Cys, Ser, Ala, The, Arg, Pro, His, Glu, Val, Ile, Met, Asp, Trp, Tyr, Phe
Ketogenic amino acids
Broken down into acetyl-coA, can’t synthesise glucose
Ile, Lys, Leu, Tyr, Trp, Phe
Removal of alpha amino group in aa breakdown
Transfer of NH2 to alpha keto acid
Aspartame + alpha keto glutarate —> OAA + glutamate
Alanine + alpha keto glutarate —> pyruvate + glutamate
Catalysed by aminotransferases with pyridoxal phosphate cofactor
Occurs in liver
Reversible
Removal of alpha amino group from glutamate
Glutamate dehydrogenase reaction
In mitochondrial matrix
Glutamate oxidised to schifi base intermediate then hydrolysed to alpha keto glutarate and NH4+
Oxidative deamination reaction
NH4+ —> urea, driving reaction forwards
Urea cycle net reaction
Co2 + NH3 + 3ATP + Aspartate + 2H2O —> urea + 2ADP + Pi + AMP + PPi + fumarate
Urea cycle linked to TCA cycle by fumarate
Control of urea cycle
Carbamoyl phosphate synthetase 1 (CPS1) activated allosterically by N-acetyl glutamate
N-acetyl glutamate synthesised by reaction of glutamate and acetyl-coA
Enzyme synthesising this is activated by arginine
Arg is an intermediate in urea cycle so if its conc increases not enough carbamoyl is entering the cycle
Role of glutamine in nitrogen metabolism
Some ammonium needed for synthesis of nucleotides and aas
Glutamine acts as non toxic carrier of nitrogen and can be used as a nitrogen donor
Glutamine synthase:
Glutamate + NH4+ + ATP —> glutamine + ADP + Pi + H+
Glutamine neutral so can be moved in blood
Ketone body synthesis
2 acetyl-coA —> acetoacetate —> beta hydroxybutyrate / acetone
Occurs in liver mitochondrial matrix
Ketone body degradation
Beta-hydroxybutyrate —> acetoacetate —> 2 acetyl-coA
1st step reduces NAD+
Glyoxalate cycle
Can produce glucose precursors with acetyl-coA as animals can’t make glucose from acetyl-coA
Isocitrate —-> glyoxalate + succinate
By Isocitrate Lyase
Glyoxalate —> malate
By malate synthase
Malate — OAA by malate dehydrogenase
OAA —> PEP —> glucose
uses of ketone bodies
Some acetyl-coA —> ketone bodies when not enough OAA to enter TCA cycle
Alternative fuel source
Heart muscle uses ketone bodies
In starvation we get a high conc of ketone bodies
Brain can adapt to use them
Cholesterol synthesis
2 acetyl coA —> acetoacetyl-coA —-> beta hydroxyl beta methylglutaryl coA —-2NADPH—-> mevalonate —-> cholesterol
Key junctions in metabolism
Glucose-6-P
Pyruvate
Acetyl-coA
How is Pentose phosphate pathway regulated
NADP+ activates glucose-6-phosphate dehydrogenase
Acts as e- acceptor in dehydrogenase reaction
Competes with NADPH for space in the active site
Glycogen phosphorylase cofactor
PLP
