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Nobel Prizes
1907 Eduard Buchner, cell-free fermentation chem.
1915 Richard M. Willstätter, researches on plant pigments, esp. chl.
1918 Fritz Haber, synthesis of ammonia from its elements, chem.
1922 Archibald Vivian Hill, production of heat/lactic in muscle
1923 Frederick Grant Banting, discovery of insulin
1929 Arthur Harder, fermentation of sugar and fermentative enzymes
1931 Otto Heinrich Warburg, discovery of respiratory enzyme action
1953 Hans Krebs, the TCA cycle, phy.
1964 Dorothy Hodgkin, determinations by X-ray tech of the structures
of important biochemical substances, VB12,chem
1964 Konrad Bloch mechanism and regulation of cholesterol and fatty
acid metabolism, phy
1970 Luis F. Leloir, discovery of sugar nucleotides and their role in the
biosynthesis of carbohydrates, chem.
1977 Rosalyn Yalow, peptide hormone production in brain and
radioimmunoassay
1978 Peter D. Mitchell, understanding of biological energy transfer
and the chemiosmeric theory, chem
1985 Michael S. Brown, regulation of cholesterol metabolism, phy
1988 Johann Deisenhofer, determination of 3D structure of
photosynthetic reaction center
1992 Edmond Fischer, reversible protein phosphorylation
1997 Paul D. Boyer, elucidation of enzymatic mechanism in synthesis
of ATP
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Principles of Bioenergetics
ATP provides energy by GROUP TRANSFERS, not by simple hydrolysis
2+
Adenylate kinase requires Mg
Transphosphorylations between Nucleotides Occur in ALL Cell Types
INORGANIC Polyphosphate Is a Potential Phosphoryl Group Donor
Biochemical and Chemical Equations Are NOT Identical
Biological Oxidations Often Involve DEHYDROGENATION
Free-energy calculation: the STANDARD Reduction Potential
FMN contains ribulose in CHAIN form, not in circle form
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Glycolysis and the catabolism of hexose
Thiamine pyrophosphate is the coenzyme of transketolase
TPP carries the active acetaldehyde groups
The phosphofructokinase in bacteria and plants uses PPi
Aldolase: Class I: animal/plants, form Schiff base;
2+
Class II: bacteria/fungi, no Schiff, Zn instead
2+
PGAld dehydrogenase: contains His and Cys residue, inhibit by Hg
Pentose phosphate pathway: transaldolase: 7+3=4+6
transketolase: 5+5=3+7; 3+6=4+5
transaldolase/transketolase intermediate: resonance stabilization
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Regulation of Glycolysis
Glycogen phosphorylase only acts on the reducing ends of glycogen,
split α-1,4 bonds and produce G-1-P
The debranching enzyme (glucantransferase) first transfers extra
glucose chain to another reducing end, forms α-1,4 bond. Then
cleaves the α-1,6 bond and removes the remain one glucose.
In liver, the G-6-P enters ER lumen and cleaved by
glucose-6-phosphatase on membrane, forms G and Pi, add blood Gs.
The source of glycogen genesis: UDP-glucose; G6PÆG1P +UTP
ÆUDPG +Pi.
Amylo transglycosylase or glycosyl(4,6) transferase transfers glycogen
fragments from nonreducing ends to chain middle thus made α-1,6
bonds while glycogen synthase make α-1,4 bonds during glycogen
synthesis.
Glycogen synthase requires a primer with at least 8 Gs to initiate
glycogen synthesis, which synthesized by glycogenin(Tyr involved).
Hexokinase Isozymes (I-IV) of Muscle and Liver Are Affected
Differently by Their Product, Glucose-6-Phosphate.
Muscle: hexokinase I, II(predominant),III: high G affinity, inhibit by
G6P
Liver: hexokniase IV: low G affinity and Km, inhibit by specific protein
but not G-6-P.
Phosphofructokinase-1 Is under Complex Allosteric Regulation.
Pyruvate Kinase Is Allosterically Inhibited by ATP, acetyl-CoA, ala and
FAs, cAMP dependent and PKA stimulated. L form is inactive and also
regulated by hormones. In liver it was also inhibited by glucagons.
Fructose 2,6-BPi Is a Regulator of Glycolysis and Gluconeogenesis
Insulin stimulates GLUT4, hexokinase and glycogen synthase
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The Citrate Cycle
3 steps: 1)production of acetyl-CoA; 2)acetyl-CoA oxidation;
3)electron transfer and oxidative phosphorylation
The electrons removed from the hydroxyethyl group derived from
+
pyruvate pass through FAD to NAD in E3
th
FeS in aconitase: 3 Cys binds to Fe and the 4 Fe binds to the citrate
+
+
2+
2 types of isocitrate dehydrogenase: NAD /NADP , includes Mn
Succinyl-CoA synthetase: His residue transfers Pi to the GTP
4 anaplerotic pathways in calvin cycle: pyruvate Æmalate through
malic enzyme; pyruvate Æoxaloacetate through pyruvate
carboxylase; PEP to oxaloacetate through PEP carboxykinase or PEP
carboxylase
Fatty acid catabolism
Intestinal lipases degrade triacylglycerols in intestinal lumen.
Fatty acids and other breakdown products are taken up by the
intestinal mucosa and converted into triacylglycerols.
Triacylglycerols
are
incorporated,
with
cholesterol
and
apolipoproteins, into chylomicrons.
Chylomicrons move through the lymph and bloodstream to
tissues.
z Lipoprotein lipase, activated by apoC-II in the capillary,
converts triacylglycerols to fatty acids and glycerol.
z The surface of adipocytes in cell are coated with perilipins, a
family of proteins that restrict access to lipid droplets
z VB12: 5’-deoxyadenosine +Corrin ring system(CO3+)
+amino-isopropanol
+dimethyl-benzimidazole
ribonucleotide
z Fatty acids bind to albumin for the blood transportation.
z Glycerol catabolism: see atlas
z Fatty acid activation: 2 ATP consumed
„ FAs+ATPÆFA-AMP+PPi; FA-AMP+CoA-SHÆacyl-CoA+AMP
z Acyl-CoA transport through mt membranes: carnitine
acyltransferase;
‹ I: outer membranes; II: inner membranes
z Rate-limiting step for FA oxidation: carnitine transport
speed. 3 steps.
z Malonyl-CoA inhibits the carnitine acyltransferase I in order
to prevent simultaneous synthesis and degradation of FAs.
z FA oxidation has 3 steps: β-oxidation, TCA, electron transfer
& oxidative phosphorylation
z When TFP(bound to mt membranes) has shortened the fatty
acyl chain to 12 or fewer carbons, further oxidations are
catalyzed by a set of four soluble enzymes in the matrix.
z The phosphorylation of ACC made it lost the activity to
synthesize FAs.
Glucagen and PKA stimulate
phosphorylation while insulin inhibits.
z The β-oxidation enzymes (short-chain specified) in mt and
+
G bacteria have 4 separate subunits; the G bacteria has
one enzyme in whole; mt long-chain specified has Enz-1
separated and 234 in whole; peroxisome and glyoxysomal
has Enz 1/4 separated and others form MFP.
z The ω oxidation occurs in the ER of liver and kidney, minor
way, acts while β oxidation is defective.
z The α oxidation deals with branched fatty acids.
z Animals can convert odd-number FAs to glucose only, while
plants can both even and odd
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Amino acids catabolism
Pyridoxal phosphate (PLP, VB6) is the cofactor of
transaminase
+
Glutamate release its amino group as NH4 in liver
Alanine transports ammonia from skeletal muscle to the liver
Activity of urea cycle is regulated on 2 levels.
1.
Acetyl-CoA+ GluÆ N-Acetylglutamate (arginine stimulates);
2. N-Acetylglutamate stimulates the synthesis of
carbamoyl-Pi.
Each urea cycle costs 3ATP, leaves 2ADP and 1 AMP and 4 Pi
H4-folate and adoMet transfers one-carbon units.
The sulfur atom in adoMet carries a positive charge
VB12 deficiencies can be cured by either add VB12 and also
folate
Glycine degradation would lead to 3 ways: carbon to serine
and produce N5, N10-methylene-H4-folate; by H4-folate to
CO2; to glyoxylate and finally oxalate.
Branched-chain amino acids (Ile,Leu,Val) are not degraded
in the liver but in peripheral organs as muscles, adipose,
kidney and brain.
Oxidative phosphorylation and photophorylation
Different cytochromes have different hemes. a: Heme A
(CHO+pentene); b: Iron protoporphyrin IX; c: Heme c
(CH3CH-S-Cys)
Electron transfer
NADHÆ(X)rotenoneÆQÆcytb(X)antimycinAÆcytc1ÆcytcÆ
cyt(a+a3)Æ(X)CO,CNÆO2
Electron transfer complexes
Complex I: NADH to ubiquinone; NADH dehydrogenase;
+
contains Fe-S and FMN; catalysis 1) NADH+H +QÆ
+
+
+
+
NAD +QH2, 2)NADH+5H N+QÆNAD +QH2+4H P
Complex II: Succinate to ubiquinone; succinate
dehydrogenase; membrane bound, contains heme b and Q
site, 2Fe-S, FAD. (as cytb, cytc1)
Complex III: Ubiquinone to Cytochrome c; contains cytb,
cytc1, 2Fe-2S, heme bH/bL/c1, binds free cytc,
+
+
QH2+2cytc1(oxidized)+2H NÆQ+2cytc1(reduced)+4H P
Complex IV: Cytochrome c to O2; cytochrome oxidase; 2CuA
& 1 CuB, 2Fe-2S, heme a/a3, binds free cytc, 4 cytc (reduced)
+
+
+ 8H N+O2Æ4 cytc(oxidized)+4H P+2H2O. (as cyta, cyta3)
All hemes bound to cyt tightly, however, only heme C
covalently bond.
Alternative mechanism for oxidizing NADH in plant
mitochondria:
+
+
2Gly+NAD ÆSer+CO2+NH3+NADH+H
Chemical uncouplers of oxidative/photo phosphorylation:
+
+
DNP and FCCP, provide dissociable H and carry H across
the inner mt membrane and dissipate the proton gradient
mt F1 ATP synthase: α3 β3 γ δ ε, β has ATP catalytic activity.
mt F0 ATP synthase: a b2 c10-12
+
each ATP requires 4 H to formation
+
for every 3 ATP synthesized, 10-14 H required
malate shuttle: aspartate(penetrable) +α-ketoglutarate Æ
glutamate+oxaloacetate(later
malate,
penetrable),NADHÆNADH
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liver, kidney and heart
glycerol 3-phosphate shuttle: glycerol 3-phosphate Æ dihydroxyacetone phosphate, NADHÆFADH2, skeletal muscle and brain
Heat was generated by uncoupled mitochondria in brown fats.
Chlorophyll funnels the absorbed energy to reaction centers by
exciton transfer
Purple bacteria: P870,PQ(II); Green sulfur bacteria: P840,Fe-S(I)
+
H (from H2O) Æ P680ÆP680*Æ PheoÆ PQA(plastoquinone)
nd
ÆPQB(2 quinine)Æcyt b6fÆ Plastocyanin ÆP700 ÆP700*
ÆA0(electron acceptor chl) ÆA1(phylloquinone) ÆFe-S ÆFd
+
+
ÆFd:NADP oxidoreductase ÆNADP
Light Harvesting Complex (LHC): chl a+chl b+ lutein
+
+
+
2H2O+2NADP + ~3ADP+8 H ÆO2+2NADPH+ ~3ATP+2H ,
+
+
+
2+
PS II: 4 P680+4H +2PQB+4H Æ4P680 +2PQBH2, has 4 Mn
+
+
+
PS I: 2Fd(red)+2H +NADP Æ2Fd(ox) +NADPH +H ,produce
reduced ferredoxin.
Concerning PSI requires less energy than PSII, in order to
prevent exciton larceny, the PSI and PSII were separated
spatially. The PSII located in the stack of thylakoids while PSI
facing out.
Cyt b6f complex links PSII to PSI, contains b-type cyt and 2
+
hemes (bH/bL) Rieske Fe-S, cytf, pumps H from stroma to
thylakoid lumen
Plastocyanin was free in chloroplasts as cytc in mitochondrial
2+
In P680, the Mn binds to Tyr has 5 oxidative status.
Red drop: chloroplast efficiency drop when over 680nm
Red lights made chloroplast reduced and far-red made it oxidized.
Carbohydrate synthesis
Fixation of CO2 has 3 stages: fixation; reduction; regeneration of
acceptor
Rubisco activase: dissociate RuBP from the Lys residues of
2+
rubisco , Lys interacts with carbomoyl and Mg thus activate it.
3-PGAÆ1,3-DPGA: 3-PGA kinase, requires ATP
1,3-DPGAÆPGAld: 3-PGA degydrogenase, requires NADPH
PGAldÆdihydroxyacetone phosphate:triose phosphate isomerase
PGAld+Dihydroxyacetone phosphate ÆF1,6BP: transaldolase
F1,6BPÆF6P: F1,6 bisphosphatase (FBPase-1)
F6PÆstarch (ct matrix)/sucrose(cytosol)
Each triose phosphate from CO2 requires 6 NADPH and 9 ATP,
meantime, each glucose requires 12 NADPH and 18 ATP
Glycogen synthase and glycogen phosphorylase are reciprocally
regulated.
When glycogen synthase dephosphorylated, it
activates(a); glycogen phosphorylase dephosphorylated, it
inhibited(b). And visa versa.
Lack of rubisco, sedoheptulose 1,7-BPase, Ru5P kinase, animals
can not synthesize CO2 into glucose.
4 calvin cycle enzymes were indirectly regulated by light: Ru5P
kinase,FBPase-1,
sedoheptulose
1,7-BPase,PGAld
dehydrogenase. Activated while 2Cys S-S bond cleaved by
ferredoxin(light energy)
2+
Light, high pH and high [Mg ] activates FBPase-1
C4 photosynthesis: pyruvate+ATP ÆPEP(+AMP+PPi) Æ(+CO2)
Æoxaloacetate(+Pi) Æ(+NADPH)Æ malate (enter bundle
sheath) Æpyruvate +CO2+NADPH
ADP-Glucose is the substrate for starch synthesis in plant and for
glycogen synthesis in bacteria
Starch(n)+G1P+ATPÆstarch(n+1)+ADP+2Pi
UDP-Glucose is the substrate for sucrose synthesis in the cytosol
of leaf cells.
F6P+UDP-glucoseÆsucrose
F6PÆ(PFK-2)ÆF2,6BP; F2,6BPÆ(FBPase-2)ÆF6P
PFK-2 was activated by Pi and inhibited by 3-PGA
FBPase-1 was inhibited by F2,6BP
Sucrose 6-phosphate synthase: when phosphorylated, less active
Lactose synthesis: UDP-galactose and glucose
UDP glucose: intermediate of glucuronate and VC
Cellulose synthesis: initiated by lipid-linked primer, UDP-glucose
Lipid Biosynthesis
Malonyl-CoA comes from acetyl-CoA and CO2, irreversible,
acetyl-CoA carboxylase
Acetyl-CoA carboxylase in bacteria has 3 separate subunits;
animal has a single MFP; plants have both. Contain biotin.
ACP: contains 4’-phosphopantetheine
The CO2 formed during condensation process is the same CO2
that has been added to acetyl-CoA and generates malonyl-CoA.
The dehydration process formed a trans double bond in FA syn.
Fatty acid synthase complex: ACP; KS (β-ketoacyl-ACP-synthase);
MT (Malonyl-CoA-ACP transferase); KR (β-ketoacyl-ACP
reductase); HD (β-hydroxyacyl-ACP dehydratase); ER (enoyl-ACP
recuctase); AT (acetyl-CoA-ACP transacetylase)
The main FA synthesis process forms palmitate (16 C)
Each added malonyl-CoA requires 2 NADPH
In photosynthetic cells of plants the FA synthesis go in ct stroma
For FA synthesis, the acetyl-CoA were shuttled out from
mitochondria as the form of citrate. Citrate, pyruvate and
malate can pass through mt membranes, and citrate was formed
by oxaloacetate and acetyl-CoA.
Animal FA synthesis rate-limiting step was acetyl-CoA
carboxylase, which covalently regulated by citrate binding and
phosphorylation. Phosphorylation was triggered on by glucagon
and epinephrine. Citrate binding activates the process while
glucaton/epinephrine, palmitoyl-CoA inhibit.
Plant/bacteria acetyl-CoA carboxylase was stimulated by
2+
increased pH and [Mg ]
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Long-chain saturated FAs are synthesized from palmitate on ER
Mammals can not synthesize linoleate 18:2(∆9,12) and
α-linolenate (∆9,12,15)
Eicosanoids are formed form 20-carbon polyunsaturated FAs
arachidonase by cyclooxygenases and peroxidases
Triacylglycerols and glycerophospholipids are synthesized from
the same precursors: acyl-CoA and L-glycerol-3-phosphate.
First the diacylglycerol-3-phosphate (phosphatidic acid) was
formed, then to triacylglycerols/glycerophospholipids.
Release of triacylglycerol stimulated by glucagons/epinephrine
Insulin stimulates the synthesis of fatty acids and acetyl-CoA.
Glycerol-3-phosphate was formed form dihydroxyacetone
phosphate through glycerol-3-phosphate dehydrogenase.
The rate-limit step of glyceroneogenesis was PEP carboxylase
2 strategies in the forming of phosphodiester bond of
phospholipids: 1) diacylglycerol activated with CDP; 2) head
group activated with CDP. Prokaryote can use 1) only.
PhosphaditylserineÆphosphadityethanolamineÆphosphatidycho
-line, the CH3 in choline was donated by adoMet.
Plasmalogens contain ester(double bond) -linked R group,
oxidized by mixed-function oxidase.
The condensation of palmitoyl-CoA and serine produce
β-ketosphiganine, then sphinganines. Then double bond and R
group was introduced. Finally ceramide, cerebroside (Glu) and
sphingomyelin (choline)
Isoprene was the basic of cholesterol. All carbons by acetyl-CoA.
Synthesis of cholesterol has 4 steps: 1)acetateÆmevalonate
(NADPH); 2)mevalonateÆactivated isoprene; 3)activated
isoprene to squalene(NADPH);4)squalene to cholesterol(NADPH).
LDL was most rich in cholesterols while HDL has the least.
LDL enters the cell by endocytosis.
Biosynthesis of amino acids, NAcs and other.
Each N2 fixation requires 16 ATP
Nitrogenase complex contains: 4Fe-4S, Iron-molybdenum
cofactor (1Mo, 7Fe, 9S, 1homocitrate).
Glutamine synthease is the primary regulatory in N metabolism.
When Gln synthease subunits adenylylated, activity goes low.
PII-UMP stimulates deadenylylation.
Cys residue played role in Gln amidotransferase, 2 domains.
222SO4 ÆAPSÆPAPSÆPAPÆSO3 ÆS ÆCys
Chorismate is a key intermediate to synthesis Trp, Phe & Tyr.
Amino acid biosynthesis is under allosteric regulation.
Concerted inhibition: the overall effect is more than additive.
Glycine is a precursor of porphyrins.
Heme is the source of bile pigments.
δ-Aminolevulinate is the precursor of porphyrins, its synthesis
requires tRNA-Glu.
Jaundice: leakage of bilirubin.
Glutathione peroxidase contains Se.
Arginine is the precursor of biosynthesis of NO.
Aspartate transcarbamoylace under allosteric regulation of C/ATP.
The salvage pathway of NAc: alkaline base +PRPPÆNMP+PPi
Metabolism regulations and hormones
Radioimmunoassay (RIA):
calculate the [bound(radiolabeled)/unbound] value to determine
the hormone amount in unknown samples.
nd
NO: cytosolic receptor (guanylate cyclase) & 2 messenger cGMP
Cori cycle: lactate generated in skeletal muscles were transferred
into liver and been recycled as glucose.
Neurons can use keto bodies (β-hydroxybutyrate)
The pancreas secretes insulin or glucagon to response blood G.
The starvation leads to the high ketone concentrations in blood.
Insulin activates the GLUT4 glucose transporters on membrane.
Acetone come from spontaneous decarboxylation of acetoacetate
Diabetes, accumulation of acetyl-CoA, leads to the
overproduction of acetoacetate and β-hydroxybutyrate.
Leptin was produced in adipocytes and acts on hypothalamus to
curtail(reduce) appetite; stimulates production of anorexigenic
hormones; regulates gene expression. Defective causes obesity.
Adiponectin acts through AMP-dependent kinase (AMPK).
Defective leads less sensitive to insulin.
Phosphorylated ACC(acetyl-CoA carboxylace) is inactive, AMPK
phosphorylates ACC.
Enzymes use NAD as cofactor:
Isocitrate dehydrogenase; α-ketoglutarate dehudrogenase; G-6-P
dehydrogenase; Malate dehydrogenase; Glu dehydrogenase (use
either NAD or NADP); glyceraldehydes-3-phosphate (PGAld)
dehydrogenase; lactate dehydrogenase; alcohol dehydrogenase
Enzymes use FAD as cofactor
Fatty acyl-CoA dehydrogenase; dihydrolipoyl dehydrogenase;
succinate dehydrogenase; thioredoxin reductase
Enzymes use FMN as cofactor
NADH dehydrogenase (Complex I); glycolate dehydrogenase
Diseases
Deficiencies in carnitine: inability to transport FAs, hemodialysis,
organic aciduria, weakness
CPT I: affect liver and reduce FA oxidation and ketogenesis
CPT II: recurrent muscle pain, fatigue and myoglobinuria
MCAD deficiency: vomit, lethargy & coma, prevent fasting
Refsum’s Disease: inherited disorder, lack mt α-oxidizing enzyme,
phytanic accumulation, cerebellar ataxia, nerve deafness.
LHON: defects in mitochondria encoded cytb gene.