The binding of N-(phosphonacetyl)-L-aspartate to aspartate carbamoyltransferase of Escherchia Coli

Vol.

136,

April

29,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1986

Pages

The Binding of N-(Phosphonacetyl)artate Aspadate Carbamoyltransferase of Escherchia Karl

W. Volz, Kurt

L. Krause,

and William

822-826

to Coli

N. Lipscomb

Glbbs Chemical Laboratory, Harvard University 12 Oxford Street, Cambridge. MA 02135 Received

March

10,

1986

A more precise description of the binding of N-(phosphonacetyl)Laspartate to the catalytic chains of aspartate carbamoyltransferase clarifies aspects of the specificity of this enzyme toward its substrates, carbamoylphosphate and L-aspartate, and suggests a catalytic role for His-134. 0 1986 *ca,+emic Press,

Inc.

Aspartate

carbamoyltransferase

bamoyltransferase forming

dimensional

bound

This

regulatory

R form

allosteric dimers

structures

to which

the

pyrimidine

and phosphate enzyme

consists

rs in a molecule are known

the substrate

from of

(c&(rs)s

pathway

car-

in E. coli

carbamoylphosphate

two

catalytic

of symmetry

for both the less active

analogue

Laspartate

and

trimers

cs and

De (1).

T form (l-3).

N-phosphonacetyl-Laspartate

by

Three and the

(PALA)

is

(4). We present

tural

initiates

carbamoyl-L-aspartate

Laspartate. three

EC 2.1.3.2)

(Carbamoylphosphate:

features

here some details which

may relate

of the binding

to the catalytic

of PALA and comment

on struc-

mechanism.

Method8 The three dimensional structure of the complex between aspertate carbamoyltransferase and PALA has been further refined ueing the programs of Hendrickson and Konnert (5) with conjugant gradient calculations following the method of Jack and Levitt (6). Although there is some disorder in the regulatory chains, particularly for residues l-10 and 50-50, the agreement factor, RI, for the entire structure is presently 0.23 for the 50,605 unique reflections to 2.3A. The effective resolution of this study is, however, 2.5A et which the observed reflections are 95% complete. At the present stage we have included 150 water molecules in the asymmetric unit which contains csrs. and have refined thermal parameters for individual atoms. Effectively 92% of the atoms of the protein have been included in the refinement. Essentially all of the catalytic chains and particulary the binding region for PALA are welt ordered. 0006-291X/86 Copyright All r&h/s

$1.50 0 1986 &v .4ccrdwnic Press, qf‘ reprodwtion in a,~,\’ form

Inc. reserved.

822

Vol.

136,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Remulta

The details As shown,

this

of the interactions highly

and one lysine, these because

negatively

and with

interactions

occur

from

the T to the R form

lytic

chain

also bind

within

the catalytic

change

the homotropic

binding.

These

conformational

molecule

have been described

changes

by l2A and reorient by 15” about

The phosphonate tive portlon hydrogen

moiety

to Ser-52,

and 55. Lys-84 and Ser-80

from

from

to which

twofold

with

the catalytic by 10’. while

with

c chain

of the

in substrate of the PALA trimers

move

the regulatory

axes.

It makes

and the peptide

change,

the binding

electron

salt links

cata-

subunits

both can participate

to one another

an adjacent

an adjacent

the two

but

transition

conformational

bringa

In them,

Most of

PALA binds,

from

of PALA is the most

Thr-55,

four arginines

in the homotropic

associated

the molecular

of the molecule. bonds

occurs

previously.

relative

chain

in Fig. 1.

bond donors.

An even larger

so that residues

with

of hydrogen

transition.

are shown

interacts

some residues

the PALA molecule.

together

reorient

that

of the enzyme,

chain

diiers

molecule

of number

catalytic

apart

charged

with the enzyme

a number

of a conformational

also accompanying

of PAM

rich

Arg-54

NH groups

and most

nega-

and Arg-105, of residues

and 53, 54.

also bind to this portion.

CYB 47

TY~ 240

Fxg. 1:

Biding

the holoenzyme, other methods from PAL4 to

of N-(phosphonacetyl)-Laspartate c a~0 aspartate (Cys-4’7, Lya-83.

carbamoyltransferase. Tyr-165, Lys-232

have direct interactions. 823

to the catalytic and

Residues Tyr 240)

unit of

studied are too

by far

Vol.

136,

No. 2, 1986

BIOCHEMICAL

The a-carboxylate Arg-167.

group

The P-carboxylate

in addition,

this group

of Leu-287,

His-134.

is bound

whiie

Thr-55.

the other

is bound

(adjacent

Gln-231.

bond of PALA forms

COMMUNICATIONS

chain),

Arg-105

No direct

chain,

but

bond to the carbonyl

bond

interactions

in Pig. 1, including

and

and Arg-229.

a hydrogen

the CO of the PALA peptide

shown

RESEARCH

by Lys-Q4 from the adjacent

by Arg-167.

and Arg-105.

residues

BlOPHYSlCAL

to Lys-84

is also bound

The NH of the peptide group

AND

is hydrogen

occur

Cys-47,

bonded

between

to

PALA and

Lys-Q3, Tyr-165,

Lys-232,

and Tyr-240.

Discussion The surprisingly

large

carbamoyltransferase enzyme

shows

enzyme

must

inate

against

the critical

for its substrates, select L-aspartate

by CHe in place the bond

ably shift

if present, through

is missing

It is likely

this This

and discrimthat

binding

and the POs group

is linked

resembles

supplies

could occur

either

directly

water

molecule.

from

a nearby

which

to the peptide state

negative

as the NH0 of aspartate base by accepting 824

residue,

carbonyl in which

conceiv-

donation

to

Such donation,

such as His-134,

bonded

this carbon.

either

or

to the CO of

on the carbonyl

attacks

a proton,

could

proton

phosphate.

charge

of car-

has not departed.

is hydrogen

gen of carbamoyl

also act as a general

with

the NH2 group

and obscure

a developing

phosphate

of compounds

unit, however,

in carbamoyl

role for His-134,

PALA

but the phosphate

elsewhere,

is present

between

an intermediate

of the phosphonate group

occur

the binding

PALA. is that it may stabilize

134 could

that

and L-aspartate.

that

Although

which

catalytic

of specificity

the f3-carboxylate

intermediates.

P oxygen

A possible

favors

CO and NH has formed

an intervening

and aspartate

(310&(7),

interactions

site

of 0. PALA otherwise

a potential

PAM

phosphate

at a low concentration

the active

of the CHg group

the bridging

carbamoyl

acids.

to reaction

between

The presence

to the high degree

of the many

is that

phosphate

between

for this discrimination.

interpretation

some resemblance bamoyl

ammo

interactions

and the enzyme

of interactions

may be related

all other

Another

number

directly

oxyHisor via

Vol.

136,

No. 2. 1986

a water other

molecule, obvious

His-134 phate

BIOCHEMICAL

from

would

for a general

enhance

BIOPHYSICAL

RESEARCH

as NH: an L-aspartate

this group

candidates

AND

base within

its polarizing

COMMUNICATIONS

is bound.

the active

There

are no

site. Protonation

effect on the CO group

of

of carbamoyl

phos-

(5). Another

aspect

of the structure

binding

of reaction

intermediates

Arg-105

and Lys-54.

Both bridge

group

of PALA.

natural

If binding

substrates

carbamoyl might

question

gested

by Collins

interactions

and Stark

residues

then

these

‘bridging’

residues

residues,

would

to react. process

(9), it may be reasonable

but there

to favor

of PALA indicates

a position

model

appear

and the a-carboxylate

of the catalytic

and one threonine

the participation

group moieties

into

aspects

“compression”

of the PALA molecule, suggest

bound,

and L-aspartate

with their

Two serine

substrate

would

of two

the phosphonate

to both

the mechanical

that

is the presence

are similarly

phosphate

of the R form

to identify

that

help

bring

Although

we

originally

sug-

these

specific

to the phosphonate

group

(10). are bound

is no biochemical

of an intermediate

evidence

to our knowledge

phosphoserine

to

or phosphothreonine

in the mechanism. Finally, same trimer movement

of active

the sharing

sites between

may aid in substrate of lysines

binding

two catalytic

and product

release.

53 and 54 in and out of the active

moves along the T to R continuum,

may help in opening

chains

sites,

within

the

In particular, as the enzyme

up the active

site.

Acknowledgments We thank

the National

research.

We also thank

collection

Resource

(Grant

Institutes

of Health,

N. h. Xuong,

for Crystallography

R. Hamlin

Grant

GM06920

for support

and C. Nielson

at the University

of this

for use of the data

of California,

San Diego

RR01644).

Reference3

1. Monaco,

H. L., Crawford,

J. L and Lipscomb,

W. N. (1978)

PTOC. N&L

AC&

see

USA 75, 52704200. 2.Honzatko. R. B., Crawford, J. L. Monaco, H. L.. Ladner, J. E.. Edwards, B. F. P.. Evans, D. R., Warren, S. G., Wiley, D. C.. Ladner. R C. and Lipscomb, W. N. (1982) J. Mol. Biol. 160.219-263.

825

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BIOCHEMICAL

3. Ke. H.-M., Honzatko. USA 81. 4037-4040.

AND

BIOPHYSICAL

R. B., and Lipscomb.

4. Krause, K. L., Volz, K. I. and Lipscomb, 62, 18431647.

RESEARCH

W. N. (1964) W. N. (1985)

hoc

Proc.

COMMUNICATIONS

N&J. Acad

Nat. Acad

S&.

S& [IsA

6. Hendrickson, W. A., and Konnert. J. (1981) In Riomolecular S&u&w-s, Funotion. Con,formcrtion, and Evolution (Srinivasan, R., ed.) vol. 1, pp. 4347, Pergamon Press, London. 6. Jack, A. and Levitt, 7. Christopherson, 85.

M. (1978) Acta Crystallogr.

R. 1. and Finch.

L. R. (1977)

6. Roberts, hf. F., Opella, S. J., Schaffer, (1976) J. EM. Chem. 251, 59765965. 9. Collins,

K. D. and Stark,

G. R. (1971)

Sect. A34, 931-935. Biochim.

M. H., Phillips.

J. Biol.

Cheer.

10. Jacobson, C. R. and Stark, G. R. In The Enzymes, P. D., ed.) vol. 9, pp. 225-306.

826

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Acat 481, 80-

H. M. and Stark,

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246. 6599-6605. Third

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