Download LipidCat+AAmetabolism

Lipid Catabolism,
concluded;
amino acid metabolism
Andy Howard
Introductory Biochemistry
22 April 2008
What we’ll cover
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Lipids, concluded
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Similarities &
differences
Trafficking
Bookkeeping
Special Cases
Locations for synthesis
Regulation by
hormones
Absorption and
mobilization
Lipoproteins
Ketone bodies
Lipid & Amino Acid Metabolism
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Amino acid
metabolism
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Nitrogen sources
Nitrogenase
Essential aa’s
Transaminations
General categories
Specific pathways
p. 2 of 44
22 April 2008
Similarities and differences
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… between synthesis and oxidation of
fatty acids:
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Acetyl CoA  Malonyl ACP  fatty acid
Fatty acid  FA CoA  acetyl CoA
Text gives clear exposition: read it!
This could easily result in a final exam
question!
Lipid & Amino Acid Metabolism
p. 3 of 44
22 April 2008
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Moving acyl CoA
into the
mitochondrial matrix
Carnitine shuttle moves them:
Fatty acyl CoA gets into the
intermembrane space
Carnitine acyltransferase II acylates
carnitine
Acylcarnitine crosses inner
membrane
Similar enzyme inside gets acyl CoA
back
Lipid & Amino Acid Metabolism
p. 4 of 44
Carnitine:
3-hydroxy-4trimethylammoniobutanoate
22 April 2008
Bookkeeping
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Remember:
mitochondrial or bacterial FA oxidation
yields one NADH and one QH2 per pair of
carbons, e.g. for stearate (C18):
Stearoyl CoA + 8HS-CoA + 8Q + 8NAD+
 9 acetyl CoA + 8QH2 + 8NADH +8H+
That adds up to 12+20+90-2 = 120 ATP!
Lipid & Amino Acid Metabolism
p. 5 of 44
22 April 2008
Comparisons with glucose
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Glucose is 6 carbons, not 18, so the 120 ATPs
vs. 32 comes out more like 120 vs.96 on a percarbon basis…
But per gram:
1 g * 32 mol ATP (mol glucose) / 180 g/mol =
32/180 = 0.178 moles ATP per gram glucose
1 g * 120 mol ATP /(mol stearate) / 288 g/mol) =
120/288 = 0.417 moles ATP per gram stearate
Hydration matters too!
Lipid & Amino Acid Metabolism
p. 6 of 44
22 April 2008
Efficiency of fatty acyl synthesis
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8 acetyl CoA  8 malonyl ACP = 8 ATP
8 syntheses * 5 NADPH/synthesis = 40
9 Acetyl CoA * 17 ATP/AcCoA = 153
Total: 201 ATP
So efficiency = 120/201 = 0.597
That’s a typical efficiency defined as
(energy derived from oxidation) /
(energy required for synthesis)
Lipid & Amino Acid Metabolism
p. 7 of 44
22 April 2008
Odd-chain
fatty acids
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Methylmalonyl
CoA
Rarer than even-chain but they do exist
Broken down as with even-chains but with
priopionyl CoA as end-product
Condenses with bicarbonate to form Dmethylmalonyl CoA
Racemized to L-methylmalonyl CoA
Mutated to succinoyl CoA via an
adenosylcobalamin-dependent reaction
This can actually be a source of sugars!
Lipid & Amino Acid Metabolism
p. 8 of 44
22 April 2008
Catabolism of cisunsaturated fatty acids
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Normal beta-oxidation until we encounter a
double bond
Double bond moves from cis-3,4 to trans-2,3 via
3,2-enoyl-CoA isomerase reaction
Further beta oxidation proceeds until we
encounter the next double bond;
Cis double bonds at even positions get modified
by 2,4-dienoyl-CoA reductase from trans,cis-2,4
to trans-3
3,2-enoyl-CoA isomerase moves trans-3 to
trans-2 and then we can -oxidize again
Lipid & Amino Acid Metabolism
p. 9 of 44
22 April 2008
Regulation I
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Key hormones:
insulin, glucagon, ephinephrine
Under low-glucose conditions:
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glucagon and ephinephrine circulate at high
concentrations
-oxidation encouraged
Glucose not needed for fuel so it’s conserved
High glucose conditions:
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insulin, glucagon & ephinephrine ,
FA synthesis dominates
Glucose used as fuel for making fatty acids
Lipid & Amino Acid Metabolism
p. 10 of 44
22 April 2008
Regulation II
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Main regulatory enzyme:
acetyl-CoA carboxylase
High insulin levels after meal
stimulates formation of malonyl
CoA
Product allosterically inhibits
carnitine acyltransferase so FAs
stay in cytosol
Lipid & Amino Acid Metabolism
p. 11 of 44
Carnitine
palmitoyl transferase
PDB 2RCU
144 kDa
dimer
Monomer
shown
22 April 2008
Mobilization of
fatty acids
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Triacylglycerols transported through circulatory
system in lipoprotein masses (cholesterol +
various MW proteins forming shell around lipid)
Lipoproteins hydrolyzed via lipoprotein lipase
extracellularly
Fatty acids & glycerol released extracellularly,
FAs re-esterified
What happens next depends on needs:
Triacylglycerols hydrolyzed to FAs and
monoacylglycerols, and sometimes further
High [insulin] inhibits hydrolysis
Lipid & Amino Acid Metabolism
p. 12 of 44
22 April 2008
Glycerol and
free fatty acids
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Glucagon
PDB 1GCN
3.3kDa monomer
Some of them diffuse through the adipocyte
plasma membrane & enter blood
Glycerol metabolized in liver to (…) glucose
FAs travel bound to serum albumin to heart,
skeletal muscle, & liver—
energy source esp. in fasting
Glucagon  means inhibition of acetyl CoA
carboxylase, so less malonyl CoA made
Meanwhile: high [acetyl CoA],[NADH] means
inhibition of pyruvate dehydrogenase
Lipid & Amino Acid Metabolism
p. 13 of 44
22 April 2008
Absorption of
lipids from food
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Majority of dietary lipids are
triacylglyerols;
Smaller amounts of phospholipids &
cholesterol
Suspended fat particles are coated
with bile salts, amphipathic
cholesterol derivatives
Pancreatic lipase secreted into small
intestine degrades triglycerides (in
fat particles) at C-1&3
Colipase helps bind the lipase to its
substrates
Lipid & Amino Acid Metabolism
p. 14 of 44
Lipase-colipase
complex
PDB 1LPB
10.4 kDa+ 50kDa
heterodimer
22 April 2008
What bile
salts do
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Taurocholate,
a bile salt
Bile-salt micelles travel to intestinal wall
Monoacylglycerols & free FAs are
absorbed and bile salts are released
Bile salts recirculate rapidly
When fully formed triglycerides are
made, those travel via chylomicrons for
transport to other tissues
Lipid & Amino Acid Metabolism
p. 15 of 44
22 April 2008
Dietary
phospholipids
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Phospholipase A2 in intestine
hydrolyzes ester bond at C2; the
resulting lysophosphoglycerides get
re-esterified in the intestine
High [lysophosphoglyceride] disrupts
membranes: that’s how snake
venoms work on erythrocytes
Lipid & Amino Acid Metabolism
p. 16 of 44
PDB 1G4I
13.5 kDa
monomer
Bovine
pancreas
22 April 2008
Dietary
cholesterol
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Cholesterol esters are
hydrolyzed in the lumen of the
intestine
Free cholesterol is solubilized by
bile-salts for absorption
Free cholesterol often esterified
in the intestine to form choesteryl
esters
Lipid & Amino Acid Metabolism
p. 17 of 44
Cholesterol
esterase
PDB 2BCE
64 kDa
monomer
Bovine
22 April 2008
Lipoproteins
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Core
Spherical vehicles for
transport of fats
Several sizes
Biggest, least dense:
chylomicrons
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found in blood only after a meal
Cartoon courtesy
Deliver triacylglycerol & cholesterol to U. Wisconsin
muscle and adipose tissue
Stevens Point
Remaining cholesterol-rich particles
deliver cholesterol to liver
Contains Apolipoprotein E - binds to
specific receptor in liver cells
 Others are smaller,
Lipid & Amino Acid Metabolism
more dense
p. 18 of 44
22 April 2008
Types of lipoproteins
(cf. table 16.1 & fig. 16.30)
Type
Chylo- VLDLs
IDLs LDLs
microns
MW*10-6
>400 10-80
5-10
2.3
, g cm-3
<0.95 <1.006 <1.019 <1.063
Composition (%)
Protein
2
10
18
25
Triacylglycerol 85
50
31
10
Cholesterol
4
22
29
45
Phospholipid
9
18
22
20
Lipid & Amino Acid Metabolism
p. 19 of 44
HDLs
.18-.36
<1.21
33
8
30
29
22 April 2008
% protein and density
1.12
Density depends on % protein
1.1
HDLs
1.08
Density, g cm-3
1.06
1.04
LDLs
1.02
IDLs
1
0.98
VLDLs
0.96
0.94
0.92
Chylomicrons
% protein
0.9
0
5
10
Lipid & Amino Acid Metabolism
15
20
p. 20 of 44
25
30
35
22 April 2008
Protein components
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Structural amiphathic crust
proteins:
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ApoB-100 (513 kDa) bound to outer
layer of VLDLs, IDLs, LDLs.
ApoB-48 (241 kDa): N-terminal end
of ApoB-100, found in chylomicrons
Smaller, less strongly bound
proteins
Some are responsible for specific
binding to receptors in cells
Lipid & Amino Acid Metabolism
p. 21 of 44
Kringle
domain of
ApoA1
PDB 3KIV
8.7 kDa
monomer
Human
22 April 2008
Low-density lipoproteins
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LDLs deliver cholesterol to peripheral tissues via
cell-surface binding
High intracellular [cholesterol] inhibits synthesis
of HMGCoA reductase and the receptor
People without LDL receptor: cholesterol
accumulates in the blood and gets deposited in
skin and arteries
This risk leads to the description of LDLs as
“bad cholesterol”
Lipid & Amino Acid Metabolism
p. 22 of 44
22 April 2008
High-density lipoproteins
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Take cholesterol out of plasma and return it
to the liver
Binds to receptor SR-B1 and transfer
cholesterol & cholesterol esters back to liver
cells
Lipid-depleted HDLs return to plasma
Because these tend to deplete cholesterol
from the bloodstream, they become known
as “good cholesterol”
Lipid & Amino Acid Metabolism
p. 23 of 44
22 April 2008
Serum
albumin
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Free fatty acids carried
by this protein
7 binding sites for Fas
HSA also binds many
hydrophobic drugs
Lipid & Amino Acid Metabolism
HSA + 7 palmitates
PDB 1E7H
64 kDa monomer
p. 24 of 44
22 April 2008
acetone
Ketone bodies
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Three compounds produced as
stored-fuel molecules
-hydroxybutyrate &
acetoacetate are fuel
Serve as water-soluble lipids—
readily transported in plasma
Important in brain, skeletal
muscle, intestine during
starvation
Lipid & Amino Acid Metabolism
p. 25 of 44
acetoacetate
hydroxybutyrate
22 April 2008
Synthesis of
ketone bodies
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Starts out like steroids:
2 acetyl CoA 
acetoacetyl CoA  HMG CoA
Then HMG CoA lyase converts HMG
CoA to acetoacetate and acetyl CoA
Acetoacetate can be reduced via
NADH to -hydroxybutyrate
Acetoacetate can also be
nonezymatically decarboxylated to
acetone
Lipid & Amino Acid Metabolism
p. 26 of 44
HMGCoA lyase
PDB 2CW6
EC 4.1.3.4
197 kDa hexamer
human
22 April 2008
Oxidation of
ketone bodies
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-hydroxybutyrate oxidized back to
acetoacetate in a separate version
of the liver enzyme that made it
acetoacetate converted to
acetoacetyl CoA in mitochondria in
nonhepatic tissues via succinyl-CoA
transferase
Thiolase converts acetoacetyl CoA
into two molecules of acetyl CoA
Lipid & Amino Acid Metabolism
p. 27 of 44
Succinyl CoA
transferase
PDB 1OOY
212 kDa
tetramer
dimer shown
pig heart
22 April 2008
Amino acid metabolism
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As Horton says, this is a difficult subject
to cover
Hundreds of reactions, dozens of
reaction pathways
Some common threads and
generalizations
We’ll focus on the latter
Lipid & Amino Acid Metabolism
p. 28 of 44
22 April 2008
The nitrogen pool (fig. 17.1)
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Nitrogen fixation from air (N2  NH3)
doesn’t produce a large percentage of
circulating biological nitrogen but it’s the
ultimate source of most of it
Other entries in pool: nitrate (NO3 -),
nitrite (NO2-)
Most of this difficult biochemistry is
bacterial
Lipid & Amino Acid Metabolism
p. 29 of 44
22 April 2008
Nitrogenase
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Enzyme found in Rhizobium, a bacterium
that colonizes & lives symbiotically in the
root nodules of legumes and a few other
plants
Also in free-living microorganisms like
Azotobacter
Energetically expensive but irreversible
path to reduction of dinitrogen to
ammonia:
N2 + 8H+ + 8e- + 16 ATP 
2NH3 + H2 + 16ADP + 16Pi
Lipid & Amino Acid Metabolism
p. 30 of 44
22 April 2008
Structural features
of nitrogenase
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Multi-component complex
Mo-Fe active site in actual N2fixing component
Nitrogenase
Probably proceeds via diimine and Mo-Fe + Fe
proteins
hydrazine:
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N=N +
+
 H-N=N-H
H-N=N-H + 2e- + 2H+  H2N-NH2
H2N-NH2 + 2e- + 2H+  2 NH3
2e-
2H+
Lipid & Amino Acid Metabolism
p. 31 of 44
PDB 1G20
350 kDa
heterooctamer
Azotobacter
22 April 2008
Ammonia,
nitrate, nitrite
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Ammonia comes from decayed
organisms and is oxidized in soil
bacteria to nitrate (nitrification)
Nitrate reductase and nitrite
reductase found in plants and
microorganisms:
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NO3- + 2e- + 2H+  NO2- + H2O
NO2- + 6e- + 7H+  NH3 + 2 H2O
Lipid & Amino Acid Metabolism
p. 32 of 44
Nitrate
reductase
PDB 2BO0
111 kDa
trimer
monomer
shown
Alcaligines
22 April 2008
Essential and non-essential
amino acids
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An amino acid is defined as essential if it
must be obtained within the diet
In general the essential amino acids are
the ones that have complicated and
highly ATP-dependent biosynthetic
pathways
Of course, it depends on the organism
Lipid & Amino Acid Metabolism
p. 33 of 44
22 April 2008
The human list
(cf. box 17.3)
AA
moles ATP
Asp
Asn
Lys
Met
Thr
Ala
Val
Leu
Ile
21
22-24
50-51
44
31
20
39
47
55
essential?
no
no
yes
yes
yes
no
yes
yes
yes
Glu
Gln
30
31
no
no
Lipid & Amino Acid Metabolism
AA
moles ATP
Arg
Pro
Ser
Gly
Cys
Phe
Tyr
Trp
His
44
39
18
12
19
65
62
78
42
p. 34 of 44
essential?
no
no
no
no
no
yes
no*
yes
yes
22 April 2008
Transaminations
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General process of interconverting amino acids and -ketoacids
Primary way that N gets incorporated
into non-N-containing structures
Lipid & Amino Acid Metabolism
p. 35 of 44
22 April 2008
Reaction dynamics
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All (?) transaminations involve PLP
as a cofactor: see mechanism, fig.
7.18
These are actually oxidationreduction reactions, since we’re
swapping an amine (carbon
oxidation state +2) for a carbonyl
(carbon oxidation state 0)
But there is no external oxidizing
agent
Lipid & Amino Acid Metabolism
p. 36 of 44
Aspartate
aminotransferase
PDB 2Q7W
87 kDa dimer;
Monomer shown
E.coli
22 April 2008
Examples of transaminases
Reactants
Products
Keto acid
amino acid
Pyruvate
glutamate
Pyruvate
aspartate
Oxaloacetate glutamate
3-P-OH-pyr glutamate
3-OH-phenyl- glutamate
pyruvate
keto acid
-k-glutarate
oxaloacetate
-k-glutarate
-k-glutarate
a-k-glutarate
Lipid & Amino Acid Metabolism
Transaminase
amino acid
alanine pyruvate
alanine pyruvate
aspartate aspartate
P-ser
phosphoserine
tyrosine tyrosine
p. 37 of 44
22 April 2008
Catabolic or anabolic?
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From the point of view of available pools
of amino acids, these are ambiphibolic:
They involve synthesis of one amino acid
at the expense of another
Lipid & Amino Acid Metabolism
p. 38 of 44
22 April 2008
Biosynthetic pathways to
specific amino acids
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Some are complex
and energy-requiring
Can be logically
divided according to
chemical properties
of the target amino
acids:
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Lipid & Amino Acid Metabolism
Small
Branched-chain
aliphatic
Neutral polar
Acidic
Basic
Aromatic
Sulfur-containing
p. 39 of 44
22 April 2008
Glutamate
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Glutamate is a critical
metabolite because so
many of the
transaminations start with
it as the amine donor
It is produced in E.coli, etc.
via glutamate
dehydrogenase using
ammonium ion as nitrogen
donor:
-ketoglutarate + NH4+ +
NAD(P)H + H+  NAD(P)+
+ H2O + glutamate
Lipid & Amino Acid Metabolism
p. 40 of 44
1BGV
296 kDa hexamer
monomer shown
Clostridium
22 April 2008
Glutamine
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Glutamate can be aminated
with expenditure of ATP to
form glutamine:
glutamate + NH4+ + ATP 
glutamine + ADP + Pi
Note that glutamine
synthetase is a ligase: the
ATP is an energy-provider,
not a phosphate donor
Lipid & Amino Acid Metabolism
p. 41 of 44
Glutamine
synthetase
PDB 2OJW
211 kDa pentamer
human
22 April 2008
Aspartate and
asparagine
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Asp is simple:
transamination of oxaloacetate
Asn is straightforward too
asparagine synthetase moves
the amine from gln to asp,
leaving glu (another lyase)
Gln + asp + ATP 
AMP + PPi + glu + asn
Lipid & Amino Acid Metabolism
p. 42 of 44
Asparagine
synthetase B
PDB 1CT9
243 kDa
tetramer
E.coli
22 April 2008
Simple: ala,
gly, ser
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Alanine by transamination from
pyruvate
Glycine from serine by SHMT (q.v.)
Serine from 3-phosphoglycerate:
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Phosphoserine
phosphatase
3-phosphoglycerate + NAD 
PDB 1NNL
NADH + H+ + 349 kDa dimer
phosphohydroxypyruvate
human
3-phosphohydroxypyruvate + glutamate
 3-phosphoserine + -ketoglutarate
3-phosphoserine + H2O  serine + Pi
Lipid & Amino Acid Metabolism
p. 43 of 44
22 April 2008
Serine
hydroxymethyltransferase
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Serine + tetrahydrofolate 
PDB 2DKJ
H2O + glycine + 5,10-methylene- 90 kDa dimer
Thermus
tetrahydrofolate
thermophilus
This can be viewed as a source of
methylene units for other
biosyntheses
PLP-dependent reaction
Lipid & Amino Acid Metabolism
p. 44 of 44
22 April 2008