Vol. 171, No. 3, 1990 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

28, 1990

Pages

Allosteric

Control of Quaternary States in 23. coli Aspartate Transcarbamylase Raymond Gibbs

Received

1312-1318

August

C. Stevens

and William

N. Lipscombt

Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138

14,

1990

Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when 4TP binds. When the R-state is produced by binding of phosphonoacetamide and malonate, addition of CTP or CTP+UTP decreases the separation of catalytic trimers by 0.5 8. In the unliganded Glu239-+Gln mutant, in which the T-state is destabilized so that the enzyme exists in an intermediate quaternary state, ligation of ATP transforms the mutant enzyme to the R-state, whereas CTP converts this enzyme to the T-state. Thus; this mutant is much more sensitive t.o heterotropic allosteric control than is the native enzyme. In this communication we propose a preliminary model based on new crystallographic results that heterotropic regulation occurs partly through control of the quaternary structure by these effecters, thus regulating catalysis. @1990 Academic

Press,

Inc.

Aspartate

transcarbamylase

ferase) from Escherichia

cola is one of the most widely

catalyzes

the first committed

phosphate

and L-aspartate

tivated

by the product

Poised

at the beginning

regulation

to feedback

of the parallel

biosynthesis;

inhibition purine

of the pyrimidine

the flux of metabolites

tein crystallography the catalytic

step in pyrimidine

is not as well understood. on the regulatory

the catalytic

chain produce

How does the binding

opposite

effects on the enzyme’s

whom correspondence

0006-291XBO Copyright All rights

should

be addressed.

$1.50

8 1990 by Academic Press, Inc. of reproduction in any form reserved.

1312

(CTP)

activity?

and ac(ATP;

2).

enzyme Pro-

valuable

into

On the contrary, of ATP

(1).

blocks of DNA.

and CTP

chain at sites more than 60 a from the nearest

controlled?

To

(3).

carbamoyl

this cooperative

and have provided

ATCase

phosphate

5’-triphosphate

pathway,

mutagenesis

cooperativity

between

5’-triphosphate

form the building

and homotropic

enzymes.

the reaction

adenosine

biosynthetic

carbamoyltrans-

and inorganic

by cytidine pathway,

allosteric

that eventually

and site-directed

mechanism

local region

t

studied

to form N-carbamoyl-L-aspartate

The enzyme is susceptible

helps control

(E.C. 2.1.3.2; also called ATC ase, aspartate

insight

heterotropic to the same active site on

And, how is catalysis

Vol.

171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS CTP + UTP complexed T and R-state ATCase. T-state crystals were grown in 100 mM maleic acid, 10 mM Tria HCl, pH 5.7. R-state crystals were grown in 20 mM phosphonoacetamide (PAM), 20 mM malonate, pH 5.8. 1 mM CTP and 1 mM UTP were soaked into the crystals at pH 7.0. 179,967 reflections were collected on the CTP+UTP T-state enzyme. These data were reduced to 41,272 independent reflections to 2.4 di with an Rmrrgc = 7.1%. The crystallographic parameters are: space group P321, a = b - 122.2 A, c = 142.0 A. 213,846 reflections were collected at the Resource for Crystallography at the University of California, San Diego on the CTP+UTP complexed R-st,ate ATCase. These data were reduced to 46,733 independent reflections to 2.3 A with an Lcrgc = 7.1%. The crystallographic parameters are: space group - P321, a = b =: 122.2 A, c = 155.0 A. Both data sets were collected with three different crystals. The program X-PLOR (A. T. Btinger, X-PLOR Manual, version 1.5 (1988)) was used in all refinements. Molecular replacement was used in both structure solutions. Presently, the structures have been refined to 2.4 A resolution and to crystallographic R-values of 0.17 and 0.18 for the T- and R-states, respectively. A complete crystal structure analysis will be published elsewhere. Preliminary crystallographic studies of Gln239 ATCase with effectors ATP or CTP. Screened x-ray precession photographs were taken from crystals grown in 45 mM maleic acid, 45 mM Tris Base, pH 5.8 buffer with 10 mM ATP or 2 mM CTP. The crystallographic parameters for the ATP liganded Gln239 enzyme are: a = b = 122 A, c = 156.0 A. The crystallographic parameters for the CTP liganded Gln239 enzyme are: a - b = 122 A, c = 142 A. All enzyme complexes are crystallized in the space group P321. Conformational differences between enzyme with various substrate/effecter complexes. Con&rm&nal changes were determined by first superimposing the a-carbon atoms of one catalytic trimer and then by superpositioning the o-carbon atoms of the other catalytic trimer. The movement of the latter catalytic trimer along the three-fold axis with respect to the first catalytic trimer is the catalytic trimer separation. The unliganded enzyme was used as a reference for the T-state, while the PAM+malonate complex provided a reference for the R-state. These small changes in separation of catalytic trimers are statistically significant because the errors in position of an entire polypeptide chain are much smaller than the errors (o) associated with individual atomic positions. The approximate standard error of the mean for the separation of catalytic trimers is u/h which is about 0.01 A for the n = 1860 C, atoms in two catalytic trimers and for the 0.5 A value of u for an individual C, position based on the Luzzati plot (4). Since the standard error of the catalytic trimer separation also depends on the cell dimension along the c-axis, the unit cell dimensions were determined on at least three different crystals. The average unit-cell dimensions were used in the refinement of the crystal structure.

RESULTSAND DISCUSSION Both

the T and R-states

of ATCase

crystallize

in the space group

unit cell axes are a = b = 122 A, c = 142 A. The R-state 122 A, c = 156 A. Th e separation c-axis cell length When PAM

unliganded

T-state

and malonate

crystals

identical

(a substrate are produced

(c-axis

trimers

structures.

which

can be determined

T-state

and subsaturating

have a c-axis unit-cell

trimers

= 142 A) are soaked in the substrate into its R-state

conformation

dimension 1313

crystals are complexed amounts

of L-aspartate

of 149 A, intermediate

the

are a = b =

by monitoring

the catalytic

of 156 A (5). S oa k’m g an d co-crystallization

When unligated

for the reverse reaction)

In the T-state,

unit cell dimensions

axis along which

the single crystal is transformed

crystal which has a c-axis cell length produce

of catalytic

(th e c-axis is the three-fold

P321.

the move).

analogues as a single experiment5

with phosphate single crystals between

the

Vol.

171, No. 3, 1990

T and R c-axis intermediate

BIOCHEMICAL

cell length

crystal refined

relatively

small fraction

of total scattering

A). The T-state

complexes

triphosphate)

structures

by 0.4 A toward

the R-state.

an 11 A increase in the separation

of catalytic ATP

CTP decreases the separation

binding

bisubstrate analogues

trimers, effect

in both the unliganded

the crystal

that CTP and UTP

structure,

very slightly. with

CTP

In contrast.

Transformation X-ray

tion of Glu239 the T-state

ATP.

Glu239

scattering

occurs with with

In the pAR5

mutant, with

scattering

residues

the neutral

is intermediate

residue

chains

involved

is Asp236

enzyme

(16).

This

region

chain.

1314

destabilizing

enzyme

yielded

a

of 147 A for with

the c-axis length by

of the carboxylate

of

to serine an intermediate enzyme

of the regulatory

occurs

near

the

interacts

interactions Asp236

chain have

cl...r4

interface.

the structure

In the presence

to that shown by the wild-type

interface

of the catalytic

in

muta-

Tyr165.

the T and R forms,

similar

in several

(~4) as found

that in the absence of ligands,

between

Glu239

have been confirmed

of the wild-type

at the C terminus

curves demonstrate

between

of 156 A when complexed

is mutated

Glu239

only

when complexed

thereby

of t,his Gln239

to the mutant

Tyr165

(11). In

trimers

here that the c-axis length

the uncharged

145-153

enzyme is in the R-state,

A specific

chain

One of the interactions

(15); probably,

six new residues

mutant

(14).

liganded

The site specific

these interactions,

length

of CTP

of the T-state.

interactions

value of 142 A. These results

Lys164 than with

Solution

regulatory

CTP is added

When

is not found

in the R-state

strurture

trimers

here refine-

in ATCase

of catalytic

the T-state.

to the R-state

from solutions

Tyr165.

been replaced

the mutant

inhibitor

catalysis

of an adjacent

to disrupt

trimers

dimer about

concentrations

separation

stabilize

crystal

enzyme is increased

form of the enzyme

of the pAR5

was expected

147 A to the T-state

X-ray

X-ray

and Tyr165

involves

of catalytic

0.6 A in the direction

the T and R states (13). We report

the allosteric

more strongly

Lys164

(PALA)

and malonate

and PAM-malonate

inhibit trimers

analogue

(10). We report

The strong non-covalent

The th ree-dimensional

Gln239

decreases from low-angle

enzymes. with

to glutamine

(12).

When

of Patalytic

increases

of the catalytic

equimolar

T-state

the T-state

studies from this laboratory

between

the mutant

perturbs

the separation

chain (cl)

with

synergistically

decreases by approximately of mutant

diffraction

structure

CTP+IJTP

and UTP

of one catalytic

complexed

trimers

PAM

the separation

on

(8), and PAM

of the regulatory

the T-state

and UTP (uridine

triphosphate)

resolu-

This transformation

a 12” rotation

by 0.5 A toward

enzyme

It is known

for

liganded

substrate

ment to 2.4 A on the wild-type R-state.

are known

at medium

of catalytic

conformation.

has little

the

(7).

determined

about the 3-fold axis, and 15” rotations

the a-fold axes. In the R-state, while

of the tight

the R-state

neglecting

that have been refined are the unliganded,

(9) or weak binding

(5) in the absence of effecters induces to one another

transcarbamylase

the

form has been

structures

with ATP, the separation

Ligation

transforms

reflections

Three-dimensional

CTP ( c yt i d ine triphosphate)

(5). Up on ligation

N-phosphonoacetyl-L-aspartate

relative

in the superlattice

of ATCase

liganded,

L-aspartate

of the intermediate

of 0.25 to 5.5 & resolution

enzyme.

forms of E. COC aspartate

(adenosine

RESEARCH COMMUNICATIONS

with

structure

R-factor

of the wild-type

several enzymatic

liganded

the enzyme

The crystal

to a crystallographic

Transformation

ATP

Saturating

to its R form.

partially

tion (2.6-2.8

(6).

AND EIOPHYSICAL

between makes

of PALA,

enzyme the catalytic

several

(16). and

c...r contacts

Vol.

171, No. 3, 1990

including

a cl...r4

BIOCHEMICAL

interaction

Asp236

has been mutated

mutant

enzyme

pAR5

mutants

and confirms

conducted

the mutant

conclusion

with

confirm

conformation

a tendency

to attribute

amino

An example

contributions

Controversy

in ATCase.

One model

qua.ternary

perturbing

structure

t,he T-+R

an alteration

in substrate

transition

or high affinity sulfinate

mutations

(3) and other

affinity for substrate.

ATCase Our struc-

enzymes

can be very

there are thermodynamic

the effecters

analyses effectors

(19). regulate

cause an alteration

the activity

substrate

affinity

effects in ATCase

is cross-linked

or no homotropic

in the

of the enzyme in turn controls

by ATP

state.

substrate

analogues

by the

cooperativity

through

site-directed

into either

but at least part of the

substrate

analogues

L-cysteine

enzyme do not elicit homotropic and inhibited

The uncoupling (24).

by CTP; through

If th e wild-type

and heterotropic use of site-directed

enzyme

remains

in the alter the

affinity by perturbing

effects on ATCase

and hybridization

cooper-

here, the dominant

of homotropic

that the effecters alter the substrate

mutagenic

the low

experiments.

has been ac-

Replacement

of

chain

activation

an enzyme

some distiuc-

effects are present? then the effecters must somehow

We propose

of the regulatory

provides

chemically

studies of aspartate

a quaternary state, as observed in the wild-type enzyme. The uncoupling of ATP activation and CTP inhibition

produces

and

enzyme.

of mutant

in structural

regulating

their

enzyme,

in the wild-type

parameters

has also been observed in other instances

state and heterotropic

ATP

the earlier

changes in a mutant

study of the Gln239

considered

(24) to th e wild-type

occurs in a T-like

in ATCase

minimal

with

Furthermore,

conformational

where

the altered

(22). Binding

but the enzyme is still activated

complished

are in accord

hemoglobin

that

the enzyme

state one finds little

interactions

Lys56

velocity converts

and wild-type

and heterotropic

When

effects remain

part of catalysis

T-like

sedimentation show that ATP

mutant

thereby

affinity;

of homotropic

(23) and L-alanosine

ativity,

mutations,

(20). A n a It erna.tive model suggests tha.t the effectars ca.use

binding

the models.

heterotropic

the Lys143dAla

(21).

The uncoupling tion between

proposes

are present

and Ala236

exists over the way that heterotropic

of ATCase

equilibrium

which

interactions.

for this behavior

that are not ordinarily

control.

and

that this is an oversimplification.

between

also occurs in mutant

from residues

Allosteric

mutant

changes in kinetic

acid, we believe

studies suggest that comparisons

The

changes the enzyme to the T-state

from the crystallographic

we have demonstrated

in a single

and CTP

of interface global

(17).

of the Gln239

reported pAR5,

Difference

ATP

while CTP

effectors promote

our conclusions

it is tempting

to changes complex.

have recently the Gln239,

effecters

the importance

that the heterotropic

Although

T-R

contacts

conformation.

the allosteric

above regarding

more important,

average

interface

The results of this study of the Ala143

studies reported

catalysis

of cl...r4

and co-workers

enzyme to its R-state

conformation.

tural

to that

chain (18). Resembling

at pH 7.0 (5).

interactions

similar

the importance

Schachman

of the enzyme

conformation

enzyme exists in an intermediate

experiments

ATCase

in the T-form

to test its role in interface

an intermediate

of the regulatory

the Ala143

Lys143

to alanine

displays

only in the T form. mutation

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

with alanine produces an enzyme with CTP inhibition and chain with alanine (25). R e pl acement of Lys60 of the regulatory ATP

activation

and minimal 1315

CTP

inhibition

(26).

IJnlike

.4T-

Vol. 171, No. 3, 1990

BIOCHEMICAL

Case from E. coli, ATCase to a lesser extent

than

from

ATP).

AND BIOPHYSICAL

S. marcescens

Hybridization

experiments

E. coli is substituted

in place of the regulatory

which

characteristics

has catalytic

marcescens which

ATCase

contribute

T-R transition, ATP

(27).

site through

in reference

It appears

and cl:r4

quaternary

state.

the affinity thereby

likely

that

slightly

and co-workers

and cl:r4 interfaces

to the quaternary

binding.

Control

catalysis

a two-state

exist.

R quaternary Finally,

velocity X-ray

Although the MWC tenets is “Two (at We still do not know

and X-ray

the T*R

particularly

ATP

acces-

Schachman

proteins

and CTP

The

possess limited

in effector

has been altered

inter-

control,

conformation”

changes towards

varying

or

or intermediate

“intermediate

the T or

degrees of stability.

do not violate

induce

in the

two- or three-dimensional

differences

a signal through

and

if there are multiple

conformational

states exist, with

how the effecters transmit that

does

is similiar

the effects (18).

the MWC

is often referred to as the “two-state model”, states are reversibly accessible to allosteric

but we have determined

CTP

or CTP

scattering

or possibly

we use the phrase

states of allosteric two)

to

state, it is

of reversibly

of ATP

solution

equilibrium

that the effecters induce

model

the T+R

above,

answered.

upon complexation

diffraction,

In this communication

least

enzyme

(28) wit,h modifications

of the number

can detect such subtle

states; thus intermediate

or

is not observed

This model

and co-workers

state differences,

which

regulates

CTP

are not able to detect and describe

of whether

intermediate

binding

Although

site

separation

where the ‘l? and R states are in equilibrium

shift is so slight

Probably

are the main methods

conformations

chain,

ratio

to resolve small quaternary

based on our observation

Thus,

contacts

state in the wild-type

the T-state).

The question

model

sedimentation

the question

interface

trimer

states has not been unambiguously

enzyme that their methods

to answer

(These

(As we have noted

alter

by Her&

results.

conformational

conformations.

site (27).

to alter the R quaternary

states

does slightly

model proposed

wild-type

mediate

in the

signals from the effector

catalytic

of substrate

is not observed

the two allosteric

and, ATP

propose

of S.

signals from the effector

the enzyme’s

at the active site.

state and ATP

that the [T]/[R]

NMR,

alters

transmit

changes

speculate

ability

and CTP

“minor”

of substrate

of difference

and the effector binding

chain to the cl:rl

our new crystallographic

techniques

interactions,

state by transmitting

they do stabilize

sible quaternary

an enzyme

8).

to the primary/secondary reflecting

from

characteristics

are those that have been implicated

which

alter the R-state

(CTP

dimer

produces

and regulatory

the quaternary

regulating

alter the T quaternary

and CTP

the regulatory

of S. marcescens

interfaces

The

ATP

of the amino acid sequence reveals that the residues

differences

from these studies that ATP

to the cl:rl control

control

the regulatory

are illustrated

transition

A comparison

or cl:r4 interface

and CTP partly

dimer

by both where

of E. coli ATCase

to the regulatory cl:rl

is activated

RESEARCH COMMUNICATIONS

(29).

one of the basic oligomers” (29).

the regulatory

global

model

conformational

polypeptide changes

in the quaternary state which in turn controls substrate binding affinity and the T-+R transition. A number of questions remain to be answered but progress has been made in the understanding

of heterotropic

regulation

in aspartate

1316

transcarbamylase.

Vol. 171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This

work

fellowship

was supported

area detector

allowed

for rapid

GM06920

refinement

(WNL)

of the University

for data collection

in the T and R states, the Pittsburg which

grant

P ro f essor N. Xuong

(RCS). We thank

for use of an x-ray

by NIH

and an NIH

postdoctoral

of California

at San Diego

of the CTP+UTP

Supercomputing

complexed

enzyme

Center for the use of the Cray Y-MP

of the crystal structures,

and HURAY

for ail comparison

calculations.

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Allosteric control of quaternary states in E. coli aspartate transcarbamylase.

Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when ATP binds...
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