Anabolism of Nitrogen
Compounds
Nitrogen Cycle
Fig. 22-1 Lehninger POB 4th Ed.
Nitrogenase is a Very Important
Enzyme
.
Nitrogenase Complex
N N 2 NH4
2 H+ H2 16 ATP
4 CoA 16 ADP
4 Pyr 4 Acetyl CoA + 16 Pi
4 CO2
.
• Fe:S complexes as seen before
• Mo:Fe complexes
• Mg2+ cofactor
• doesn’t occur in aerobic environment: plant produces
leghemoglobin
Movement of NH4+
• Just like catabolism, distribution is through Glu
and Gln
• Glu not synthesized de novo in mammals,
instead KG is aminated via glutamate
dehydrogenase, a mitochondrial enzyme.
.
CO2 CO2
C O Glutamate dehydrogenase H3N C H
CH2 CH2
CH2 NH4 H2O CH2
CO2 NADPH NADP+ CO2
+ H+
.
Glutamine Synthetase is Control
Point for Nitrogen Anabolism
.
CO2 CO2
H3N C H Gln Synthetase H3N C H
+ NH4 CH2 + H 2O
CH2 Mg2+
CH2 CH2
ATP ADP
CO2 + Pi C
O NH2
.
Heavily regulated: allosteric inhibition by carbamoyl
phosphate, Trp, AMP, CTP, His, Gly, Ala,
glucosamine-6-phosphate
2nd Mechanism of Glutamine Synthetase
Regulation Unique: AMP Attached to Tyr
Fig. 22-7(a) Lehninger POB 4th Ed.
• Adenylated form is inactive form
• Cascade leads to adenylation as shown in 22-
7(b)
Essential Amino Acids
• PVT TIM HALL
Number of Enzymes Required to
Synthesize Amino Acids
Table 30-2, Harper’s ROB, 24th Ed.
Essential Nonessential
Phe 10 Ala 1
Val 1 (7shared) Asp 1
Trp 5 (8 shared) Asn 1
Thr 6 Glu 1
Ile 8 (6 shared) Gln 1
Met 5 (4 shared) Pro 3
His 6 Ser 3
Arg 7 Gly 1
Lys 8 Cys 2
Leu 3 (7 shared) Tyr 1
Total 59 Total 15
Semiessential Amino Acid
.
CO2 CO2
Phe Hydroxylase
H3N C H H 3N C H
CH2 CH2
O2 H2O
NADH NAD
+H
OH
.
Cys Synthesis
.
CO2 CH CH2CH2SH
NH3
Homocysteine
NH3
HOCH2 CH CO2
cystathionine
ß-synthase Serine
H2O
NH3
CO2 CH CH2CH2S CH2 CH CO2
NH3 Cystathionine
.
Cys Synthesis
.
NH3
CO2 CH CH2CH2S CH2 CH CO2
NH3
Cystathionine
H2O
cystathionine
-lyase PLP
NH4
CO2 C CH2CH3 NH3
O HS CH2 CH CO2
Cys
-ketobutyrate
.
Cys Synthesis
Homocysteine Generation
.
adenylyltransferase methyltransferase
Met SAM SAH
ATP methyl methylated
PPi
acceptor acceptor
+ Pi
SAH hydrolase
homocysteine
H2O Adenosine .
Creatine Synthesis
.
H
H3N C CO2
CH2
Arg-Gly Transaminidase
CH2 (Kidney) Glycocyamine
CH2 NH2
NH2 C
NH
Gly Ornithine HN CH2CO2
C
H 2N NH2
.
Creatine Synthesis
.
NH2 Guanidoacetate Methyltransferase NH~ Pi
NH2 C (Liver) NH C
HN CH2CO2 Mg2+ N CH2CO2
Glycocyamine SAM ATP ADP SAH CH3
Creatine phosphate
.
Creatine Function
.
Creatine
phosphokinase
Creatine Phosphate Creatine
ADP ATP
.
Creatine Degradation
. H
NH~ Pi O
Nonenzymatic N
NH C C
(Muscle) NH C
N CH2CO2
N CH2
CH3 Pi + H2O
CH3
Creatine phosphate
Creatinine .
Serotonin (5HT)
. H
CO2 CO2
H3N C H H3N C H H3N C H
CH2 CH2 CH2
Trp hydroxylase HO HO
5 hydroxytryptophan
decarboxylase N
N O2 THF DHF H O N
2 PLP
H
H H
CO2 Serotonin
5-hydroxytryptophan (5-hydroxytryptamine)
.
DOPA/Dopamine
.
CO2 CO2 H
H 3N C H H 3N C H H 3N C H
CH2 CH2 CH2
Dopa
Tyr hydroxylase decarboxylase
PLP
HO HO
O2 THF DHF H 2O
OH OH CO2
OH
Tyr Dopa Dopamine
.
Questions from last time
.
H CH3 CH3
H 3N C H C O C O
CH2 NH NH
HO CH2
serotonin N-acetylase methyl transferase CH2
CH2 CH2
N HO CH3O
acetyl CoA SAM SAH
H CoASH
Serotonin N N
(5-hydroxytryptamine) H H
monoamine N-acetylserotonin melatonin
oxidase
(MAO)
O2 H
NH4 C O
excreted as conjugates
CH2
HO
N
H
5-hydroxyindole-3-acetate
.
Epinephrine
.
H H CH3
H 3N C H H3N C H H2N CH2
CH2 CH OH CH OH
Dopamine phenylethanolamine
ß-hydroxylase N-methyltransferase
Cu+2
HO vitamin C HO HO
SAM SAH
OH O2 H 2O OH OH
norepinephrine epinephrine .
Histamine
.
CO2 NH3
H3N C H Histidine CH2
CH2 decarboxylase CH2
PLP
N NH CO2 N NH
.
Polyamines
.
H NH3
H3N C CO2 CH2
CH2 ornithine CH2
decarboxylase
CH2 CH2
CH2 CH2
CO2
NH3 NH3
ornithine putrescine
.
Polyamines
.
spermidine NH3
decarboxylated synthase ( CH2 )4
SAM NH2
putrescine CH -S-A (CH2 )3
3
NH3
.
Polyamines
.
H H H
H3N C CO2 H3N C CO2 H3N C H
CH2 CH2 SAM CH2
CH2 Mg2+ CH2
Decarboxylase CH2
PLP
S ATP PPi S A S A
+ Pi CO2
CH3 CH3 CH3
Met SAM decarboxylated
SAM
.
Polyamines
.
spermine NH3
spermidine synthase ( CH2 )3
NH2
decarboxylated CH3-S-A (CH2 )4
SAM
NH2
(CH2)3
NH3
spermine .
Glutathione Synthase
.
CO2 CO2
CO2 -glutamylcysteine
H 3N C H synthetase H3N C H
CH2 + H3N C H
Mg2+ CH2
CH2 CH2SH
ATP ADP CH2
CO2 + Pi
O C CO2
H N C H
CH2SH
CO2 CO2
H 3N C H glutathione
CO2 H3N C H
CH2 synthetase
+ H 3N C H CH2
CH2 Mg2+
H CH2 O CO2
O C CO2 ATP ADP
+ Pi O C C N C H
H N C H
H N C H H H
CH2SH CH2SH
Glutathione
(GSH)
.
Heme Metabolism
(Liver Mitochondria)
Liver Mitochondria
.
CO2 CO2 CO2
-aminolevulinate -aminolevulinate
CH2 synthase CH2 synthase CH2
CH2 PyrP CH2 CH2
C S-CoA C O CO2 C O
CO2 CoASH
O CH NH3 CH2
CH2
CO2 NH3
NH3
-aminolevulinate
-amino-ß-ketoadipate
Heme
.
Heme Metabolism
(Liver Cytoplasm)
.
Liver Cytoplasm CO2
CO2 CH2
CO2
CH2 CH2
CH2 porphobininogen
synthase, AKA
CH2
2 ALA dehydratase CH
C O CH2 N
H
CH2 2 H 2O NH3
NH3 Porphobilinogen
.
Heme Metabolism
.
CH3 CH CH2
1. uroporphyrinogen 3. uroporphyrinogen HC C CH
synthase decarboxylase N
CO2 CH3 CH3
CO2 CH2 NH HN
NH4 CO2
CH2 CH2 CH2 C C CH CH2
2. uroporphyrinogen 4. coporphyrinogen CH2 N
oxidase (Mw) HC C CH
4 III cosynthase CO2
CH
CH2 N
H CO2 CH2 CH3
NH3
CH2
CO2
Protoporhyrin
Fe2+ added in mitochondria (Mw)
to form porphyrin .
Heme Metabolism
.
C H3 CH C H2
HC C CH
N
C H3 C H3
C H3 CH C H2
3+ N
N Fe
C H2 C C CH C H2 C H3 CH C H2
C H2 HC C
N
CH
HC
N
C C OH
C H3 C H3
C O2- HC C CH
N 3+ N
C H3 C H3 heme oxygenase N Fe
C H2 C H3 CH C C CH C H2
C H2 heme oxygenase N Fe2+ N (microsomal) C H2
2
N
C H2 C C CH C H2 HC C CH
C O2- (microsomal) C H2 N C O2-
HC C CH
C O2- C H2 C H3
NADP+ C H2
C H2 C H3 NADPH + H O
NADPH + 2 C O2-
NADP C H2
+ H+ +O2
+ H+ C O2-
.
Heme Metabolism
.
O O
CHCH2 CHCH2
HN HN
HN HN
CO
P P
biliverdin reductase H Conjugation
P H P
N NADPH NADP+ N
O2 Fe3+ +H+
CHCH2 CHCH2
HN HN
O O
Biliverdin IX- Bilirubin IX-
("green") (yellow)
.
Conjugation
• Bilirubin diglucuronide
– Glucuronic acid
– Added in open chain form
– Added to propyl groups
– Increases water solubility
De novo Purine Synthesis 1
.
2-
2- O3POH2C
O3POH2C O
O PRPP synthetase O O
Mg2+ O P O P O
OH
ATP AMP HO OH O O
HO OH
AMP, GMP, IMP
PRPP
-D-ribose-5-phosphate .
De novo Purine Synthesis 2
.
2-
O3POH2C 2-
O3POH2C
O NH3
O
O O
Gln:PRPP amidotransferase
O P O P O Note change
HO OH O O HO OH in conformation
H2O PPi
Gln Glu
PRPP 5'-phospho-ß-D-ribosylamine
AMP, GMP, IMP
.
De novo Purine Synthesis 3
.
2-
O3POH2C NH3 NH3
O
5'-phosphoribosyl CH2
glycinamide synthetase
O C
2+
Mg 2-
O3POH2C NH
HO OH
ATP ADP O
5'-phospho-ß-D-ribosylamine + Pi
CO2 H2O
HO OH
H3N CH2
glycinamide
ribosyl-5'-phosphate .
De novo Purine Synthesis 4
.
NH3
H
CH2
N
O C C H
CH2
2-
O3POH2C NH O
O O C
formyltransferase
NH
N10-formyl THF R5P
HO OH
-THF
glycinamide formylglycinamide
ribosyl-5'-phosphate ribosyl-5'-phosphate
.
De novo Purine Synthesis 5
.
H H
N N
C H formylglycinamidine ribosyl C H
CH2 CH2
O -5'-phosphate synthetase
O C O
Mg 2+ HN C
NH ATP ADP NH
Gln Glu
R5P + Pi R5P
formylglycinamide
ribosyl-5'-phosphate
.
De novo Purine Synthesis 6
.
H
N
N
C H aminoimidazole ribosyl-5'
CH2 N
-phosphate synthetase NH2
O
HN C Mg2+ R5P
NH ATP ADP H2O aminoimidazole
R5P + Pi ribosyl-5'-phosphate
.
De novo Purine Synthesis 7
.
N aminoimidazole CO2 N
ribosyl-5'-phosphate
N carboxylase
NH2 NH2 N
R5P CO2 R5P
aminoimidazole Note: no biotin, aminoimidazole carboxylate
ribosyl-5'-phosphate no ATP ribose-5'-phosphate
.
De novo Purine Synthesis 8
.
CO2 N CO2 O
CH N C N
succinyl carboxamide
NH2 N CH2
ribosyl-5'-phosphate synthetase
N
R5P
Mg2+ CO2 NH2
R5P
aminoimidazole carboxylate ATP ADP
ribose-5'-phosphate Asp + Pi H2O
aminoimidazole succinyl carboxamide
ribosyl-5'-phosphate
.
De novo Purine Synthesis 9
.
CO2 O O
CH N C N NH2 C N
adenylosuccinase
CH2
N NH2 N
CO2 NH2
R5P H 2O R5P
CO2
CH aminoimidazole carboxamide
aminoimidazole succinyl carboxamide CH ribosyl-5'-phosphate
ribosyl-5'-phosphate
CO2
.
De novo Purine Synthesis 10
.
O O
NH2 C N NH2 C N
formyltransferase
NH2 N O C NH N
R5P N10-formyl THF H R5P
-THF formimidoimidazole carboxamide
aminoimidazole carboxamide
ribosyl-5'-phosphate
ribosyl-5'-phosphate
.
De novo Purine Synthesis 11
.
O O
NH2 C N
IMP synthase N
N
O C NH N
N N
R5P H2O
H R5P
formimidoimidazole carboxamide
ribosyl-5'-phosphate IMP
.
De novo Purine Synthesis 12a
.
O H
N CO2 CH2 C CO2
HN
N
N adenylosuccinate synthetase
N N
R5P Mg2+ N
GTP GDP
IMP N N
Asp + Pi H2O
R5P
Adenylosuccinate
.
De novo Purine Synthesis 13a
.
H
NH2
CO2 CH2 C CO2
N
N adenylosuccinase N
N N N
N
H2O CO2 R5P
N N
CH AMP
R5P
CH
Adenylosuccinate CO2
.
De novo Purine Synthesis 12b
.
O O
N N
HN H2O IMP D.H. N
N O N N
N
R5P NAD+ NADH R5P
IMP + H+ XMP
.
De novo Purine Synthesis 13b
.
O
O
N
N N
XMP:Gln amidotransferase N
O N N
NH2 N N
R5P Gln Glu ATP AMP
R5P
+ PPi
XMP
GMP
.
De novo Pyrimidine Synthesis 1
.
carbamoyl phosphate O
synthetase II 2-
HCO3 NH3 C OPO3
carbamoylphosphate
Gln Glu
2 ATP
2 ADP
+ 1 Pi
.
De novo Pyrimidine Synthesis 2
.
O aspartate O CO2
NH3 C OPO32- transcarbamoylase
NH3 C N C H
carbamoylphosphate
H CH2
Asp Pi
CO2
CTP
N-carbamoyl aspartate
.
De novo Pyrimidine Synthesis 3
.
O
O CO2
H
NH3 C N C H dihydroorotase N
H CH2
O N CO2
CO2 H2O
H
N-carbamoyl aspartate
dihydroorotate
.
De novo Pyrimidine Synthesis 4
.
O O
H dihydroorotate D.H.* H
N N
O N CO2 NAD+ O N CO2
NADH
H + H+ H
dihydroorotate
.
De novo Pyrimidine Synthesis 5
.
. O
O H
N
H
N orotate:phosphoribosyl
O N CO2
transferase
CO2 2-
O N O3POH2C
O
H PRPP PPi
orotate
HO OH
oritidylate
(OMP)
. .
De novo Pyrimidine Synthesis 6
.
O O
H H
N N
oritidylate
O N CO2 O N
decarboxylase 2-
2- O3POH2C
O3POH2C
O O
CO2
HO OH HO OH
oritidylate UMP
(OMP)
.
De novo Pyrimidine Synthesis 7
.
O O
H H
N N
1. NMPK
O N 2. NDPK O N
2-
O3POH2C PPPOH2C
O O
2 ATP 2 ADP
HO OH HO OH
UMP UTP
.
De novo Pyrimidine Synthesis 8
.
O
NH2
H
N cytidylate synthetase H
N
O N
PPPOH2C Gln Glu ATP ADP O N
O + Pi PPPOH2C
O
HO OH
HO OH
UTP
CTP .
Loose Ends: Ribonucleotide
Reductase
.
DP N DP N
O Ribonucleotide reductase O
reduced oxidized
HO OH thioredoxin HO H
thioredoxin
dNDP
NDP thioredoxin
reductase
FAD FADH2
NADPH NADP+
+ H+
.
Loose Ends: T Synthesis
.
Ribonucleotide reductase
UDP dUDP
NADPH +
NADP
+
+H .
Loose Ends: T Synthesis
.
Phosphatase Thymidylate synthase dTMP
dUDP dUMP
H2O Pi DHF
N5,N10-methylene THF
.
Loose Ends: T Synthesis
.
1. NMPK
2. NDPK
dTMP dTTP
2 ATP 2 ADP
.
Summary of Nucleotide Synthesis
• Purines built up on ribose
– PRPP synthetase key step
– 13 enzyme activities on 3 polypeptides (to IMP)
• Pyrimidine rings built, then ribose added
– Carbamoyl phosphate synthetase II key step
– Multifunctional enzyme (mammals): all but
dihydroorotate DH (CAD)
• Salvage is important—rapid turnover of mRNA
Summary of Nucleotide Synthesis
Source of Atoms
.
NH2 O Gln
C C N10-formylTHF
N N
C N C N Gly
C C
C C C C Asp
N N N N NH2
CO2
O CO2
NH2 O
NH4
C CH3 C C
C N C N C N Asp
C C C C C C Gln
N O N O N O
N5,N10-methyleneTHF
.
Purine Salvage Occurs by Two
Mechanisms: From Bases
.
adenine
phosphoribosyl transferase
Adenine AMP
PRPP PPi
O
O N N
N N 2-O POH C N
3 2 O N
N hypoxanthine-guanine
N phosphoribosyl transferase
Hypoxanthine (HGPRT) HO OH
IMP
O O
PRPP PPi
N N N N
N N NH2 2-O POH C N N NH2
3 2 O
Guanine
HO OH
GMP
.
Purine Salvage Occurs by Two
Mechanisms: From Nucleosides
.
Adenosine Adenosine Kinase AMP
Deoxyadenosine dAMP
ATP ADP
Deoxycytidine dCMP
Deoxycytidine Kinase
Deoxyadenosine dAMP
Deoxyguanosine dGMP
ATP ADP .
Pyrimidine Salvage Occurs Only
From the Nucleosides
.
Uridine-cytidine
Uridine kinase UMP
Cytidine CMP
Mg2+
ATP ADP
deoxycytidine
kinase
deoxycytidine dCMP
Mg2+
ATP ADP
Thymidine
kinase
thymidine TMP
Mg2+
ATP ADP
.
Regulation is Complex
• PRPP pool size is a major point of de novo
synthesis
• PRPP stimulates pyrimidine synthesis
• TDP inhibits PRPP production
AMP, GMP and IMP Feedback Inhibit
Allosterically
.
R-5-P PRPP Ribosylamine
AMP/ADP AMP
GMP/GDP GMP
IMP IMP .
AMP and GMP Inhibit Their Own
Synthesis From IMP
.
AMP ATP
IMP
GMP GTP
.
Ribonucleotide Reductase is Heavily
Regulated
Fig. 22-40(a) Lehninger POB 4th Ed.
• Note dimeric protein
• Complicated
mechanism involving
free radicals
• Overall activity
controlled by
ATP/dATP
Ribonucleotide Reductase is Heavily
Regulated
Fig. 36-10, Harper’s ROB, 24th Ed.
• Note dimeric protein
• Complicated
mechanism involving
free radicals
• Overall activity
controlled by
ATP/dATP
• Allosteric control
Pyrimidine Synthetic Enzyme Gene
Expression is Also Regulated
• Multifunctional protein (dihydroorotate
dehydrogenase separate)
• coordinate repression and derepression
(will talk about later)