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Aspartate carbamoyl Transferase (ATCase) 

Manickam Sugumaran University of Massachusetts 

Boston, MA 02125 

ATCase is the key enzyme responsible for the biosynthesis of pyrimidines 

N-Carbamoyl aspartateAspartic acidCarbamoyl phosphate

+H2N COOH

COOHOP

O NH2

OO

O NH2

O NH

COOH

COOH

N

C

O

N

NH2

OHOH

OO

PO

PO

PO

O O O

O OO

Cytidine triphosphate (CTP)

ATCase

Pi

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ATCase follows ordered Bi Bi reaction Carbamoyl phosphate binds to the enzyme first. Aspartate then comes 

and attacks the carbonyl carbon of carbamoyl phosphate. This results in a tetrahedral intermediate formation. The transition state then breaks down to produce carbamoyl aspartate (which then comes out of the 

enzyme first) and inorganic phosphate. 

E - Carbamoyl Phosphate

Carbamoyl Phosphate

Carbamoyl AspartateAspartate

InorganicPhosphate

EE - Carbamoyl Phosphate + Aspartate

E - Carbamoyl Aspartate + Phosphate

E - phosphateE

Succinic acid is a competitive inhibitor (against aspartate) for the enzyme. 

N-Carbamoylaspartate

Succinic acid (competitive inhibitor)

OP

O NH2

OO

O

Aspartic acidCarbamoyl phosphate

+H2N COOH

COOH

H2N

O

N COOH

COOH

H

COOH

COOH

Pi

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Consistent with the reaction mechanism, N‐(phosphanacetyl)‐L‐aspartic acid, the bisubstrate, transition state analog of the enzyme, serves as a 

potent inhibitor for ATCase (GKi =  10‐8 M).  

Pi

N

H

HOHO

OHO

OO

O

OP H2N

OC

Transition State

N-Carbamoylaspartate

H2N NH COOH

OCOOH

OP

O NH2

OO

O

Aspartic acidCarbamoyl phosphate

+H2N COOH

COOH

Transition State analog

N-Phosphonacetyl L-aspartic acid

N

HOHO

OHO

OC

O

OP

OC

H

H

Biosynthesis of CTP 

Glutamine + 2 ATP + O=C=O ---> Glutamate + 2 ADP + Pi + Carbamoyl phosphate 1

7

8

Enzymes:1. Carbamoyl phosphate synthetase2. Aspartate transcarbamoylase3. Dihydroorotase4. Dihydroorotate dehydrogenase5. Orotate Phosphoribosyl transferase6. Orotidylate decarboxylase7. Nucleoside monophosphate kinase8. Nucleotide diphosphate kinase9. CTP synthetase

NADNADH

+ Aspartate

N-Carbamoyl aspartate2

3

4

5

6

Dihydroorotate

Orotate

Orotidylate (OMP)

Uridylate (UMP)

H2O

UDP

UTP

CTP9

PRPPPPi

CO2

ATP ADP

ADPATP

ATP Gln +Glu + ADP

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Biosynthesis of CTP and regulation (in E.Coli) 

H2N

COOHNH2

OCO2

UMP

UDP

+ 2 ATP

COOHH2N

COOH

COOHNH

CO

OHH2N

O

Carbamoyl Phosphate

Aspartate

Glutamine+

+ 2 ADP + Pi

COOHNH

CO

O

N HH

ATCase

Dihydroorotase

Dihydroorotatedehydrogenase

COOHNH

CO

O

NH

OP

NH2

O

O

O O

N-carbamoyl aspartate

Dihydrooratate

Oratate

OHOH

OOP

O OO

COOHN

CO

O

NH

PRPPPPi

Oratate Phosphoribosyl transferase

Orotidylate

Orotidylate decarboxylase

UTP

CTP

Effect of ATP (activator) and CTP (inhibitor) on ATCase 

[S] mM

v

+ CTP

+ ATP Control

Activator (ATP) decreases sigmoidity; Inhibitor (CTP) increases sigmoidity

Aspartate

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The significance of CTP/ATP regulation 

 CTP (end product inhibition) is essential to turn off the synthetic pathway when excess CTP accumulates. 

 When high concentration of  purines accumulate and energy rich environment exist, ATP activation helps to produce enough pyrimidines for DNA replication.  

ATCase can be modified by pcmb. Modified ATCase exhibits Michaelis Menten type kinetics and is not 

responsive to either ATP or CTP. 

+ ClHg COOH

Enzyme thiol p-chloro mercuribenzoate

HS

OH

SH

+

Hg COOHS

Regenerated Enzyme

SH

SO

HHg COOH

[S] mM

v

[S] mM

v

[S] mM

v

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Catalytic and regulatory sites are located on different subunits of ATCase. 

  p‐hydroxymercuribenzoate dissociates ATCase into catalytic subunits (C3) and regulatory subunits (R2).  

  C3 and R2 can be separated by centrifugation and/or ion exchange chromatography.   

  Isolated C3 shows ATCase activity, but exhibits only typical Michaelis Menten kinetics. It is also non responsible to allosteric effectors ‐ ATP and/or CTP. 

  Regulatory subunit binds to ATP and CTP but does not have any detectable ATCase activity.  

   Reconstituion of regulatory and catalytic subunits (after the removal of mercurial) generates the fully fiunctional ATCase. 

Catalytic and regulatory sites are located on different subunits of ATCase. 

  ATCase treated with p‐hydroxymercuribenzoate (pCMB) does not exhibit allosteric kinetics. This desensitization is caused by the separation of catalytic subunits (C3) and regulatory subunits (R2) by the reaction with mercurials. 

ATCase Subunits

pCMB 2

3+

C

RR

C R C C

R

CR

R

C

C

R

CR C

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Catlytic subunit C3 and regulatory subunit R2 can be separated by centrifugation and/

or ion exchange chromatography. 

Distance migrated

C6R6

R2C3

ATCase Subunits

pCMB 2

3+

C

RR

C R C C

R

CR

R

C

C

R

CR C

Native ATCase: (C6R6) Allosteric kinetics Catalytic subunit alone : (C3)  Michaelis Menten kinetics 

Regulatory subunit: (R2) No enzyme activity;  but binds to ATP or CTP.  

Regulatory Subunits bind to ATP/CTP; but no catalytic activity.

3 R R

The i

The i

The i2 C C C

ATCase

The i

The i

The i

The i

The i

The i

C

R

C R C R

R C

R

C R C

[S] mM

v

[S] mM

v

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Reconstitution of catalytic and regulatory subunits restores the fully functional ATCase 

The image cann

The image cann

The image cann

2

3 +

R

C

R

C C

The image cann

The image cann

The image cann

The image cann

The image cann

The image cann

C

R

C R

C R

R C

R

C

R C

Reconstitution

The reconstituted enzyme now shows allosteric properties.

Catalytic subunits show normal M.M. kinetics & insensitive to ATP/CTP

Subunit structural arrangement in ATCase 

Top

Bottom

Bottom

Bottom

Top

Top

Bottom

Bottom

Bottom

Top

Top

Top

ATCase has a three fold axis of symmetry

C

C

C

C

CC

RR

RR

RR

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Crystal structure of ATCase 

Ligand interaction with ATCase 

  Aspartate exhibits positive homotropic interaction. 

  ATP shows positive heterotropic interaction. 

  CTP shows negative heterotropic interaction. 

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Effects of ATP and CTP 

  The end product metabolism, CTP inhibits ATCase reaction allosterically. 

  The activator ATP competes to the same site at which CTP binds; but the results are different. While ATP enhances the affinity of ATCase for its substrates; CTP decreases the affinity.  

Allosteric effects of ATCase can be explained by concerted model 

  The T state is more closed (tight). Therefore it is poorly accessible to the substrate.  

  The R (relaxed) state is more open and allows the substrate to bind to it freely. 

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Substrate binding to one subunit causes conformational changes in 

other subunits also. 

Tight state Relaxed state

Substrate

Nitration experiment 

C(NO2)4

Native enzyme Nitrotyrosine containing modified enzyme

Nitration of native enzyme produces a modified enzyme which has yellow color. Alteration in its conformation can now be monitored in the visible specrum

OH OH

NO2

C(NO2)4

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Active site is not in direct geometric relation with  subunit interaction site. 

Reconstitution of normal catalytic trimer with nitrotyrosine containing trimer generates the dodecamer. Succinate binding to the normal catalytic trimer, causes conformation changes in the nitrotyrosine containing trimer also. This confirm that conformational changes in one subunit are communicated to the others even though the second one does not bind to the substrate or its analog.

Tight state Relaxed state

Succinate