Predicting inhibition of dehydroquinate synthase

oo4o-4020191 m-Jo+.00

Tctnahedr~~~ Vol. 47.No. 14/15. pp.2423-2432.1991 Printedin GreatBritain

PREDICTING

@ 19%Pasamon Presspk

INHIBITION OF DEHYDROQUINATE

J. W. Frost** and Charles J. Manly I*

L. T. Piehler’, J. -L. Montchamp’. 8Department

SYNTHASE

of Chemistry, Purdue University, West Lafayette, 11DowElanco, Greenfield. IN 46140

IN 47907

(Received in USA 4 September 1990) ABSTRACT: Computational molecular superimposition studies predicted that a carbaphosphonate would be a likely inhibitor of 3-dehydroquinate synthase prior to the molecule’s synthesis. Mechanistic implications of the enzyme inhibition subsequently observed are discussed.

Development substrate

of computational

analog

involved

before

its

been developed

to evaluate

the

an

focus

of

catalyzes

(Scheme The

enigma. synthase

and

insights

putative

impressive as

a

I).’

conducive

of

analog

studies possessing

into the role DHQ synthase

the prediction

important

of enzyme

goal,

given

the

inhibition time

and

superimposition

has now

of 3-dehydroquinate

synthase.

This enzyme

has been

due

unique

to

its

chemical

in

aromatic

transformation

position

which

3-dehydroquinate

successfully structural

synthase

predicted features

the

plays in catalyzing

and

conversion

(DAHP) to product 3-dehydroquinate

would

of

dehydroquinate

not, on first

its successful

of substrate

offer

3-deoxy-D-orubino-

(DHQ).

H - 00 HC

bH DHQ

2423

inspection,

prediction

H H

acid

enzyme

still an enzymological

inhibition

which

This inhibition

is

amino the

SCHEME I.

DAHP

effort

molecular

inhibition.

-+

by a

computational

research the

this attention,

to enzyme

heptulosonic acid 7-phosphate

of

for

is an

exploits

inhibitors

amount

Despite

suitable

synthesis which

consequence

superimposition

by a substrate

be considered

chemical

An approach

in synthesis.

biosynthesis

methods

L. T. PIEHLERer al.

The mechanism oxidation

originally

of the C-5

acidification

of

the

proton

the cyclic enol ether followed The

and

C-2

formation

of the bond

apparent

ability

aforementioned

and since

to C-6

multitude

8.

unmasks

carbonyl

react

C-l

relatively

groups

of the steps

contained

and enol(ate)

an

intramolecular -

40,000

each turnover

required

for

aldol

Chemists 44,000)

thereby

generating

yielding

found

intermediate

in intermediate

condensation

which

of substrate

synthase

to

reduction

conversion

or directed

which

catalyzes

of substrate

to product

oxidation

of the

of intermediate

are catalyzed

by stereochemical/structural

the

the true

Dehydroquinate

the initial

of the C-5 carbonyl

in

by the

catalyze

However,

to product.

c @.

results

have long been impressed DHQ

with

Resulting

of DAHP to DHQ has been questioned.s14

than an oxidoreductase

in the substrate

phosphate

is then reduced

alcohol at C-5 of DAHP and the subsequent remainder

of

begins

I).

A (Scheme

the C-2 carbonyl

(MW

in the conversion

for DHQ synthase

elimination

and C-2 of DHQ.

small

more

expedites

verified

of intermediate

The C-S carbonyl in

of steps during

may be nothing

largely

to the carbonyl

which

between

of

role of DHQ synthase synthase

attached

of intermediate

by ring opening

enol(ate)

proposed*

alcohol of DAHP

features

8.

The

by functional

possessed

by the

substrate.

During metabolic

this time,

work also focused

perturbations

into furan

derivatives

was of particular

DAHP analogs was to lock the pyranosyl subsequent

furan

synthesized

and

formation their

analogs &

,9-2,6-anhydro

phosphate

At the same time,

conversion ultimately

2,6-Anhydro

examined.’

not

J.g was &

organophosphonate

an

was

j.R, although

of lethal

employed

to stabilize

the a-2,6-anhydro

zb was not a substrate

the and

analogs Jg and 29

were

/9-(2,6-Anhydro-3-deoxy-D-arabino-

inhibitor

while

inhibitory.

of DAHP were also synthesized.5*6

the a-analog

as probes

of some DAHP analogs

form of DAHP analogs into place so that ring opening

7-phosphonate

and a

The strategy

DHQ synthase

7-phosphonate

urubrno-heptulopyranosid)onate phosphate

of

of DHQ synthase

of the facile

interest.

was obviated.

inhibition

heptulopyranosid)onate

on using inhibitors

Circumvention

in plants6

4

3

2a Fi = PO,H, 2b R = OPO,H,

la R=PO,H, 1 b R = OPO,H,

a-(2,6-anhydro-3-deoxy-D-

The

phosphate

2

was a

for DHQ synthase

and

Q-

DHQ synthase

B-2,6-anhydro

was inhibited

by the

much better

while inorganic

inhibitor. phosphate

was eliminated from the &analog fi. An alternate ring oxygen. without

the

Substitution analogs

have

strategy

This penalty

to stabilize

would of

remove

losing

of a ring oxygen been

documented

DAHP

analogs

the possibility

enzyme

active

with carbon

site

is to substitute

of ring opening interactions

is, however,

to lead to disappointing

due

not a bemgn enzyme

a carbon

for

and subsequent to loss of

pyranosyt formation

a hydroxyl

modiftcation

inhibitton.’

the

furan

group.

smce carbocyclic

DHQ synthase

is a

2425

Inhibition of dehydrcquinate synthase

noteworthy

exception

exceptionally therefore

good

becomes

analogs

of

with

the

work

of

inhibitor

of

DHQ

syathase.4’*C

interest.

Given

of immediate

DAI-IP

were

carbaphosphonate

4

carbaphosphonate

1.

better

might

Knowles

inhibitors

likewise

of

demonstrating

carbaphosphonate

Synthesis

of

epimeric

that the a-2.6-anhydro DHQ

synthase

be an improved

1

an 4

phosphonate

relative

inhibitor

be

carbaphosphonate

to

relative

the

to

and phosphate

B-analogs,

to the

already

epimeric synthesized

RESULTS and DISCUSSION Before

the synthesis

a-carboxylate synthase

of epimeric

organophosphonate

by the a-carboxylate

stereochemical anomer

configuration

presumably

would suggest inhibitory.

the

carbaphosphonate

provides

a

unique which

ill

the same amino conversion active

to

acid

residues

product

DHQ.

site amino

in the

inhibition

carbaphosphonate The

attached.

This

4.

in the cyclic, in acyclic prior

to

and the carboxylate,

interpreting

Structures

were

calculations used

The

quantum

in subsequent

the electrostatic

the

term.

using PM3

aldol

the

for

Sybyl

to the

mechanics

2 of

structures in

the

overlooked.

This

which

likely

groups

center

enol(ate)

p

based

differences field full

and

were

geometry

partial

atomic

calculations

using

on

submitted

charges the Sybyl

and

notably

unmasked

superimposition 4

D.

4 were

and

performed

to avoid anion

potential

calculations.

to quantum

mechanical

in

coordinate

Cartesian

optimized

force

is

same

carbaphosphonate

unsolvated

optimization

the

become

molecular epimeric

complexity

of

is, however,

and carbaphosphonate

the computational

satisfactory

the carboxylate

have

Computational between

with groups

in the epimeric

configuration

asymmetric

with DAHP

interact

I) provided

to which

stereochemical

substrate

of the functional

functional

and

can interact

during

overlays

4 carbon

of overlays

intermediate

force

derived

be

asymmetric region of acyclic intermediate

with

prediction elements

the molecule present

A, B. or c (Scheme

condensation.

energy

DAHP

should be

by different

structural

groups

various

C-2 carbonyl

to reduce

Hamiltonian

mechanically

molecular

with

the feasibility

studies

in order

conformational

relaxed

using

functional

This offending

C-2 carbonyl, and C-4/C-5 species

intermediates

intermediates

the

The

the

& and 2

confident

otherwise

Qualitative

attempted

where

for

in

in carbaphosphonate

afforded

on whether

reactive

was opposite

intramolecular

superimposition

neutral

D

unexpected.

synthase.

identified.

intermediates.

carbon

identify

might

was the carbaphosphonate

on determining

problems

space.

reactive

that

intermediate

first then

center

intermediate

then focused

the

were

impediment

asymmetric

absent

Molecular

were

None of the cyclic

main

carbon

using

stabilize

to

of DHQ

is attached

basis

inhibition

methods

would depend

Reactive

intermediates

overlays.

immediately

which

acid residues

reactive

studies

inhibition

as a

of this

Inhibition

all. quite

but not the a-analogs

enzyme

computational

to enzyme

carbaphosphonate

after

carboxylate

suited

certainly has proven to be the case with dehydroquinate Epimeric

was,

the

the potential

appraisal.

and the same asymmetric

However,

for

be justified,

required

and &

which

seemed

4 inhibition.

important

&

to

4 could

inhibition

analogs Jg and &

results

opportunity

are

analogs

by DHQ synthase

paradoxical

epimeric

enzyme

carbon

that the p-2,6-anhydro These

inhibitors

anhydro at

bound

carbaphosphonate

to achieve

field

geometries with

inclusion

were of

2426

L. T. PIhIRER et al.

TABLE 1, Calculated Energy Values (kcal) for the Low Energy Conformations and Acyclic Intermediate p Before Molecular Superimposition PM3

Sybyl intermediate

@

carbaphosphonate

4

of Carbaphosphonate

CHARMm

1.3

-243

0.89

-21.6

-423

-26.3

Calculated Energy Values (kcal) for the Low Energy Conformations and Acyclic Intermediate p After Molecular Superimposition

intermediate

B

carbaphosphonate

4

A systematic including identified

from

optimization each

search

all significant each

-223

7.14

-20.9

-398

-16.8

was performed rotatable

search.

Entries

conformation

(Sybyl

Energy

when

conformation. relax under

minimization

allowed

(Sybyl)

the selection p.

4 and intermediate

conformations through

the geometry

of

from

acyclic

Allowing geometry

and

conformations

the

PM3 energy

intermediate

all atoms

optimization

showing for

p

is

for

those

except

these

allowed

to

important

with PM3, the calculated

geometry for

was an extended

for the superimposition

relaxed

two matched

were

conformations

reference

energies

geometries

in Sybyl

field)

mechanics

reference

this

p

(Sybyl force

molecular

of low energy

in Table 1 show Sybyl, PM3, and CHARMm geometry)

cost estimated

considerably

Several low energy

In the case of intermediate

conformation.

The higher

on both carbaphosphonate

bonds.

4

CHARMm

9.1

of these conformations

molecule.

of Carbaphosphonate

PM3

Sybyl

4

under

each

conformations

relax

slightly

to the

molecular

from

method.

is reduced the

Multifit

superimposition

energy for intermediate

to

)J drops to -236

kcal from -223 kcal. The molecular both

molecules

hydroxyl oxygen with

oxygen with

bonded from

relax.

with

acyclic

acyclic

carboxylate

superimposition

to

inspection the

with the

with

carbaphosphonate efficiently

derived

of DHQ synthase

the rate of product

hydroxyl

hydroxyl

oxygen,

carboxylate C-2

acyclic

superimposition

from quinic

were

carbaphosphonate

carbaphosphonate

singly

bonded

carboxylate

of

C-5

Table

1 and

molecules

oxygen

doubly

bonded

carbaphosphonate oxygen.

Figure

is

singly As seen

1. the

small.

C-4

hydroxyl

C- 1 hydroxyl

and

two

allowing

carbaphosphonate

carbaphosphonate

oxygen,

these

while

cost

of

Epimeric

to be an inhibitor of DHQ synthase. developed

acid.?

by

The kinetics

were first reexamined.

formation

specified oxygen,

oxygen,

results

for

using Sybyl Multifit

carbonyl

intermediate’s

conformations

was subsequently

inhibition

superimpositions C-5

bonded

4 was thus predicted

A synthesis

1 was generated

intermediate’s

molecular

superimposed

pair

C-4

doubly acyclic

oxygen

of

atom

intermediate’s

intermediate’s

intermediate’s oxygen

carboxylate

attaining

The

acyclic

of Figure

reflected

time

which

both

associated

carbaphosphonate

with carbaphosphonate

In the presence dependent

diastereomers

behavior

of epimeric indicative

were

1 slow binding

carbaphosphonate of

slow

4,

binding

Inhibition of dehydroquinate

syntbase

64

(c)

EWJRE 1,

Stereoviews of the energy minimized Sybyl Nuftifit conformers of: (a) carbaphosphonate 4, (b) carbaphosphonate 4 superimposed with acyclic intermediate Q, and (c) acyclic intermediate 9,

3427

2428

L. T. PwLRR et al.

inhibition

of

inhibition

for epimeric

DHQ

and an inhibition inhibition

synthase.”

constant

were &,a I

surprisingly,

both

Subsequent

carbaphosphonate

experiments

established

4 to be k,,

K, = 7.3 x 10mQM.

1.4 x lo6 M-‘s-l,

The same kinetic

kOrr = 7.5 x IO-’

carbaphosphonates

similar

had

the

kinetic

= 1.5 x 10’ M-‘s“, parameters

I

of this

= 1.1 x lo-’

inhibition

s-l,

for carbaphosphonate

and Ki = 5.4 x 10sD M.

s-l,

constants

3 was bound and released by the enzyme much more slowly than carbaphosphonate

1

Rather epimeric

although 2,

I

I 340

300

parameters

k,

380

wavelength (MI)

FIGURE 2,

Difference spectra obtained during incubation of epimeric carbaphosphonate 3 with DHQ synthase: (a) DHQ synthase (20 PM) in assay buffer before addition of carbaphosphonate 3, referenced to baseline; (b) One minute after addition of epimeric carbaphosphonate 4 (30 pM); (c) One hour after addition of carbaphosphonate 4.

Perhaps

the

most

interesting

trademark

carbaphosphonate

2 is the enzyme-mediated

carbaphosphonate

4 and formation

this

carbaphosphonate

carbaphosphonate argue

against

carbaphosphonate

fits

2.

The similar

a

results

in

the

slow

oxidation

of enzyme-bound enzyme

diastereomeric fit.

of

active

in an observed

of

increase

site

molecular

DHQ synthase an

improvement

superimposition

before over

the molecule that

of

studies

carbaphosphonate

inhibitor.4’*c

DHQ

synthase

Oxidation

which

between

two

incubation

that

the of

in optical

predicted

was synthesized.

of

in a fashion

indicative of inhibitor oxidation and reduction of enzyme-bound The

the

inhibition

density

DHQ

is similar

whether

to that

carbaphosphonates synthase

of

might

with

at 340 nm (Figure

by

of epimeric

NADH could thus be used to evaluate

relationship

Nonetheless,

binding

epimeric

2) which

is

NAD. epimeric

Although 2, eprmeric

carbaphosphonate

its inhibrtion

would

of DHQ synthase

carbaphosphonate

3 IS still

inhibrt is not

a potent

2429

Inhibition of dehydroquinate synthase

This inhibition

inhibitor. which

might

catalyze

in the molecular the hydroxyl intermediate

the

fully

viewed

intramolecular

aldol

condensation

superimposition

group

attached

a proton

It is the

to the anomeric protonated

carbon

of

the perspective involving

thereby

base would then

back to the C-2 carbonyl

interaction

from

the

of active acyclic

the

epimeric

catalyzing be capable

used

the proton

the ring opening of stabilizing

oxygen as the intramolecular carbaphosphonate

site residues

intermediate

A basic residue (Scheme II) could remove

studies.

The resulting

c.

well as donating proceeds.

can be more

from

of cyclic

the enolate

as

aldol condensation

4 carboxylate

with

this

“aldol

catalyzing base” which is likely responsible for the observed inhibition of DHQ synthase. SCHEME II.

HE+ D

-00

e

POSH-

&o

HB+

This

catalytic

superimposition bonded the

oxygen

of with

DHQ the

asymmetric

superimposition

singly

bonded

are achieved thus

oxygens

indicate

that

taken

are the

of

of the acyclic of

with minimal

centers

carbon

is

synthase

superimposition

and the C-2 carbonyl

carboxylate

superimpositions the

view

studies

both energy

for why epimeric

other

researchers’

form

of intermediate

aqueous

solutions

views c This

of

DHQ

intermediate

has been reactive

intermediate

molecular doubly

along with the superimposition

a-carboxylate

noteworthy

that

oxygens

attached

Computational is suitably

of such to

molecular

oriented

in three

with the putative aldol catalyzing base.

carbaphosphonate

4 binds

with

In elegant

catalysis. which

the

a-carboxylate

It is especially

superimposed.

carbaphosphonate

synthesized

during

cost even when the hydroxyl

simultaneously

synthase

consideration

carbaphosphonate

molecules.

dimensional space to take advantage of binding interactions The hypothesis

into the

generates

underwent

ring

DHQ rynthase

model

intermediate opening

experiments. c

after

and subsequent

contrasts

with

a protected photolysis

in

stereospecific

2430 L.T.F%mxi~erul.

aldol

condensation

to

nonenzymatic

conditions

stereochemical

outcome

amassed

that

a

intermediate

generate led of

DAHP

&*a*e*d.a

DHQ.

to

the

the

intramolecular

phosphate These

The

observation

suggestion

that aldol

oxygen

results

led

such

enzyme

to

reaction not

that

of

DHQ

or

under

guide

evidence

elimination

proposal

occurring

catalyze

In addition,

catalyzes

the

a

may

condensation,

possibly

have

of

the

the

has been

phosphate

synthase

from

is really

a

catalytic “sheep in wolfs clothing.“‘. If DHQ synthase the

aldol

which

the

carboxylate

of

the

molecular

points

becomes

carbaphosphonate

4 so tightly.

more

synthase

intermediate

p.

the likely

Nanomolar

to formulate

play

an

optimized

Therefore,

although

binding

could

the

be gained Enzyme

inhibition

Ihe

carboxylate

of DAHP

for

interaction

with

this

inhibitors

are,

of an enzyme

role

DHQ synthase

during

by

the

epimeric

charged

to

residue. to expect

base) as to bind

after

all, not that

active

site residue aldol

may not be a catalytic

of

is important

2, it is unreasonable

intramolecular

B,

interaction

via situation

(in lieu of an aldol catalyzing

enzyme

catalytic

eliminates

to carbaphosphonate

the existence

active

of intermediate

This

a-configuration.

to explain.

site

of the C-5 carbonyl

predicts

site would be so tolerant

reasonable to

in

difficult

4 is diastereomeric

active

reduction

consideration.

superimposition

to an active

that carbaphosphonate

DHQ

from

carbaphosphonate

that the enzyme seems

role after

is removed

4 then

which

Given

base

computational

carbaphosphonate binding

plays no catalytic

catalyzing

common. which

It

enables

condensation

“wolf,”

of

the enzyme

is

probably a good deal more than a catalytic “sheep.” EXPERIMENTAL Materrals.

DAHP was synthesized

according

to the procedure

3-deoxy-D-urabtno-heptulopyranosid)onate. elaborated

by Reimer

synthase

of 3-deoxy-D-arabmo-heptulosonic

contaminant

material

(grade

V-C)

which interfered

was

obtained

was

obtained

methyl

by

the

from

Sigma.

to the procedure Lower

procedure

Dehydroquinate

of Frost et al.ll of

grades

(methyl

was used for the

acid instead of E. coli JB-5.

from E. colt RB791 (pJB14) according

was isolated NAD+

starting

from

that E. coli BJS02aroB(pKD130A)10

et aLg with the exception

whole cell synthesis purity

This

of Frost and Knowles8

NAD+

High

contained

a

with the enzyme assay.4b

Methods.

Spectrophotometric Quantitation with

of

a Bio-Rad

computer 5.3,

was

(MOPAC

desalting

Eureka through

5.0, QCPE

out on a Perkin-Elmer

relied

column

1.O (Borland

on VAX8800

acquired

carried

concentrations

PD-IO

program

were performed

assays were

protein

on Coomassie

prior

to use.

Tripos

455, J.J.P.

Associates. Stewart,

Other

F.J. Seiler,

Lambda

Least-squares

The Sybil molecular computations Quantum

3B Spectrophotometer.

binding.12

Computational

International).

and Cray2 platforms.

dye

Enzyme

curve

fitting

molecular

utilized

the

utilized

the

superimpositions

modelling

Chemistry

was desalted

system, PM3

Program

Version

Hamiltonian

Exchange)

and

CHARMm (Quanta 3.0, CHARMm 21.2. Polygen Corporation). Determtnatron

Assay

of the Rate Constant (k,,)

solutions

consisted

for Carbaphosphonate

of deionized,

glass distilled

4 Btndrng to DHQ Synthase.

water

containing

50 mM MOPS buffer

Inhibition of dchydroquinatc

at pH 7.5, cobalt(H) incubation were

removed

at timed

Product

mL). progress

chloride

at 15 Oc for

curve

were

and k are adjustable

(0.25 mM). NAD+ (0.25 mM) and DAHP

I5 min. enzyme intervals

inorganic

and

phosphate

fitted

2431

synthase

(0.05 nmol/mL)

quenched was

with

quantitated

to the equation?’

20% (w/v) by

absorbance

Only progress

parameters).

was added

curves

the -

trichtoroacetic

Ames

(0.125 - 0.5 mL) acid

a final

(0.1 - 0.225 Points

procedure.”

a - beekt (where

with

After

(0.025 - 0.2 mM).

and aliquots

t = time

velocity

much

on

the

and a, b,

less than

the

initial velocity were used. The association rate constant, k,,. was calculated from: (k - k,,rr) (1 + tDAHPI/K,)

km =

[ carbaphosphonate

using K,

= 18 x 10s8 M and a value for k,,

calculated

k,

values

did

not

change

Determinatron of the Rate Constant (k,,) Carbaphosphonate 15 “C for

with

which

the

concentration

30 min in 2 mL of a pH 7.5 solution

by the phosphate

as described

DAHP

or

below.

The

carbaphosphonate

(0.94 or 1.25 PM) were incubated

containing

MOPS (50 mM).

An aliquot (40 pL) of this solution

of DAHP (0.7 mM) in the same MOPS/Co(II)/NAD+ was followed

of

4.

/or Carbaphosphonate f Release from DHQ Synthase.

4 (3.75 PM) and DHQ synthase

(0.25 mM) and NAD+ (0.25 mM).

4 ]

was determined

production

(for

buffer

together

cobalt(H)

at

chloride

was then added to a solution

at 15 OC, and the progress of the reaction

40 mitt) as above.

Points

on the progress

curves

were fitted to the equation: absorbance = a + beekt + c. Detection of

Enzyme-bound NADH Durtng Incubatton of DHQ Synthase

A solution

of DHQ synthase

(20 PM) in 50 mM MOPS (pH 7.5) containing

(50 PM) and NAD+ (10 FM) was placed in a quartz cuvette, nm

to

solution

250 nm of

in

a spectrophotometer

carbaphosphonate

carbaphosphonate.

Scans

4 were

with Carbaphosphonate f.

was made

at

to set

the

added

to

timed

and the solution

baseline make

intervals

absorbance.

the until

enzyme there

cobaft(I1) chloride

was scanned A small

solution

30

was

further

no

from 400

aliquot CM

of

a

in

the

rise

in

absorbance at 340 nm. ACKNOWLEDGEMENT. L.T.P was supported by a fellowship provided supported by grants from the Herman Frasch Foundation and Elanco.

by Monsanto.

Work was also

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