20' Biosynthesis of pyrimidines

1
20. Biosynthesis of pyrimidines purines
2
Pyrimidine biosynthesis starts with
carbamoylphosphate
carbamoylphosphate synthetase II
HCO3-


2 ATP 2 ADP Pi
carbamoyl- phosphate
Glu
Gln
This reaction resembles the first step of the
urea cycle, except that Gln replaces NH4 as the
nitrogen donor. Its catalyzed by a cytosolic
enzyme, carbamoylphosphate synthetase II.
3
Bacterial carbamoylphosphate synthetase II has
three catalytic sites connected by a channel
1
The enzyme has two subunits. Ammonia is produced
from Gln in the small subunit, channeled to the
large subunit, and converted to carbamate at the
second active site. Carbamate moves to the third
site, where it is converted to carbamoyl
phosphate. The total length of the channel is
about 100 Å.
2
3
4
All the other atoms of the pyrimidine ring come
from aspartate
N-carbamoyl-aspartate
aspartate transcarbamoylase

Pi
H
carbamoylphosphate
Asp
H2O
N
H
dihydroorotate
orotate
5
5-phosphoribosyl-1-pyrophosphate provides the
ribose and phosphate for pyrimidine nucleotides
C
orotate
ribose-5-P ATP
PPi
5-phosphoribosyl-1-pyrophosphate (PRPP)
AMP
uridine 5-phosphate (uridylate, UMP)
CO2
orotidine 5-phosphate (orotidylate)
6
UMP is converted to UTP and then to CTP
Glutamine provides the amino group of CTP
UMP
-
2 ATP
Glu
2 ADP
Gln
uridine 5-triphosphate (UTP)
7
Bacteria regulate pyrimidine biosynthesis by
feedback inhibition of aspartate transcarbamoylase
Asp
Pi
2 ATP 2 ADP Pi
Gln HCO3-
N-carbamoylaspartate
carbamoylphosphate
aspartate transcarbamoylase
carbamoylphosphate synthetase II
-

In bacteria, the end-product of the pathway, CTP,
inhibits aspartate transcarbamoylase. ATP
counteracts this inhibition. The effect of ATP
serves to keep pyrimidine and purine biosynthesis
in balance.
8
E. coli aspartate transcarbamoylase is the
prototype of an allosteric enzyme
The enzyme is a C6R6 oligomer of separate
catalytic (C) and regulatory (R) subunits.
Substrate (Asp) binding is strongly cooperative.
Binding of CTP to the R subunits decreases the
affinity of the C subunits for Asp. ATP blocks
this effect. In the absence of the R subunits,
the C subunits are catalytically active and are
not affected by CTP.
9
E. coli aspartate transcarbamoylase has two
distinct conformational states
R2C2 unit from a crystal structure in the taut
state (no substrate). The viewpoint is rotated
90o relative to that in the previous slide.
1ezz.pdb
R2C2 unit from a crystal structure in the
relaxed state (with a bound substrate analog).
1f1b.pdb
10
In mammals, five of the steps of pyrimidine
biosynthesis occur in two multifunctional enzymes
2ATP Gln HCO3-
carbamoyl-phosphate
1
Asp
2ADP Glu Pi
2
H2O
Pi
3
N-carbamoyl-aspartate
UMP
C
NAD
orotate phosphoribosyl transferase
5
4
orotate
I wont expect you to know the names of enzymes 3
- 5
PRPP
orotidylate decarboxylase
CO2
PPi
11
Mammals regulate the formation of
carbamoylphosphate by allosteric effects of ATP
CTP in the first multifunctional enzyme
ATP
CTP

-
ATP Gln HCO3-
carbamoyl-phosphate
Asp
carbamoyl-phosphate synthetase II
ADP Glu
H2O
Pi
N-carbamoyl-aspartate
dihydroorotate
12
Experiments with radioactive tracers showed that
the purine ring is assembled from 5 different
types of building block
Glycine
CO2
Amino group of aspartate
N10-formyl-THF
N10-formyl-THF
Amide N of glutamine
(THF tetrahydrofolate)
A common pathway of 11 steps beginning with
phosphoribosyl-pyrophosphate (PRPP) and glutamine
leads to inosine monophosphate (IMP). Separate
branches then lead to AMP and GMP.
13
The purine ring is synthesized attached to
ribose-5-P
Gln
ribose-5-P ATP
1
AMP
5-phosphoribosylamine
5-phosphoribosyl-1-pyrophosphate (PRPP)
PPi Glu
ATP
Gly
ADP Pi
2
3
14
Assembly of the purine ring continues one atom at
a time
Steps 6 7 dont require biotin. ATP is required
in bacteria, but not in vertebrates, where a
single multifunctional enzyme catalyzes steps 6-8.
15
until we reach inosine monophosphate (IMP)
Steps 8 9 resemble formation and breakdown of
argininosuccinate, but produce ADP rather than
AMP PPi
9
fumarate
IMP
C
16
Branching pathways lead from IMP to AMP and GMP
AMP
I wont expect you to know the details of these
reactions.
fumarate
Asp
GDP Pi
GTP is required for synthesis of AMP ATP is
required for synthesis of GMP
GTP
ATP
AMP PPi
NAD H2O
IMP
NADH
Glu
The purine base in IMP is called hypoxanthine.
GMP
Gln
17
Purine biosynthesis is regulated by feedback
inhibition at both the first step and the branch
point
11
1
The cancer chemotherapeutic drug 6-mercaptopurine
inhibits all three regulated steps. Before
acting, it must be converted to a ribonucleotide
by reaction with PRPP.