Labeling of the active site of glutamate dehydrogenase with a photogenerated species

BIOCHEMICAL

Vol. 52, No. 4,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LABELINGOF THEACTIVE SITE OF GLUTAMATE DEHYDROGENASE WITH A PHOTOGENERATED SPECIES* BY William E. White, Jr. and K. LemoneYielding** Laboratory of Molecular Biology, University of Alabama in Birmingham, School of Medicine, Birmingham, Alabama 35294 Received

April

30,

1973 ABSTRACT

2-Azidoisopthalic acid and 5-azidoisopthalic acid were prepared from the corresponding amines through diazonium salt intermediates. Kinetic studies indicate that they are competitive inhibitors of glutamate dehydrogenase. Photolysis of the enzyme-inhibitor complex with light > 300 nm generates a nitrene which reacts irreversibly with the enzyme and exhibits fluorescence emission at 430 nm when excited by 350 nm radiation. Glutamate dehydrogenase is an example of an enzyme subject to in vitro regulation

by a variety

investigations

of small molecules.

including

of structure-function

its

Although the subject of extensive

complete covalent structure,

relationships

cannot proceed without

of the catalytic

by pyridoxal

phosphate labeling has led to the proposal that lysine 97 is in the

center,l

labeling

techniques for enzyme sites are hampered because of reactive

nature of the reagents used, with resulting

for modifying the enzyme at mutiple sites.

Thus, affinity

labeling alone also usually doesn't establish whether a particular is in the catalytic conformation.3

of the enzyme

of this residue.2

the usual non-specific potential

Inactivation

but subsequent studies have reported residual activity

complete derivitization Affinity

sites.

unambiguous

localization

catalytic

and regulatory

complete definition

site or whether it

is merely essential

We have developed two asides which exhibit

*Supported by Grant d CA-12538 from NCI. **Recipient of RCDAfrom NIGMS.

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

1129

residue

to the enzyme competitive

after

Vol. 52, No. 4, 1973

inhibition

for

photolysis

azides

with

almost

and/or

a-ketoglutarate

present

with

These

usual

affinity

the unactivated

and thereby

species

determine

the unactivated

Competition forward

studies

with

the

in locating

in

to determine

order if must

react

it

binds

the

water

of competition center.

to the

cannot

or nitrenes

with

type

in the active

labels

which

are

It

enzyme.

be done

and therefore

two ad-

can be studied

preferentially

affinity

react

insertion,

at least

enzyme kinetics

bind

any carbenes

with

site

in a straight

not

closely

cannot

bound

react

randomly

certain

have been used with antibodies;

but

they

moderate

success

have had only

systems.

AND METHODS

from water

acid

and showed

at pH = 7.3. solution

labels

for

enzymatic

5-Aminoisopthalic only

Glutamate

in glycerol, Sigma Chemical

spectra

were

grating

spectrometer.

NADH.

First,

receptor-site

the binding

success

MATERIALS

from

labels,

energy,

direct offer

On

enzyme.

Photogenerated

minor

species

reaction.

high

abstraction,

photogenerated

chemical

Second,

dehydrogenase

due to their

by hydrogen

which

to the enzyme when formed with

which,

unequivocally species

manner.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the glutamate

nitrenes

any residue

over

in

generate

cycloaddition.

vantages

is

BIOCHEMICAL

one spot

technical

upon

dehydrogenase

analyses

paper

acid

Co. and used without

studies

were

(mono sodium

performed

salt)

as the was purchased

purification.

LJV absorption

on a Beckman Model

monitored

by Midwest

twice

in 0.1 M phosphate

from Boehringer

spectra were

was recrystallized

chromatography

further

15 and infared

Kinetic

grade)

was purchased

and a-ketoglutaric

run on a Cary

Elemental

(Eastman

by following Microlab,

A-20 AOD340 of

Indianopolis,

Indiana. Preparation mmol) was dissolved HCl.

The temperature

dissolved 3.3 mmol),

of 5-azidoisopthalic in

15 ml of Me2SO containing was lowered

in 0.5 ml of water, dissolved

acid,

5-Aminoisopthalic 1 ml of water

to 5' C and NaN02 (0.225

was added.

in 0.5 ml of water,

Ten minutes was added.

1130

acid

later

(0.50

and 0.1 ml of

g, 3.3 mmol) NaN3 (0.216

The reaction

g,

was allowed

g, 2.7

Vol. 52, No. 4,. 1973

to stir

for

water

BIOCHEMICAL

about

1 hour

precipitated

(80%).

lit5

Found:

ir

16158,

250 (s = 1.0 x 104),

certain

aniline ganate.

acid

C, 53.54;

N, 20.28; (0.1

the

free

(dec): 2105S(N3),

oxidized which

acid

could

H, 3.90;

N, 7.73; (dec).

was prepared

by the method

described

for

M phosphate,

pH = 7.3)

X = 295 (shoulder)

photolysed

N, 19.99.

through

reactor

Found:

for

was used,

C, 46.39;

5(0.6

g,

H, 2.43;

mp = app 220 (dec).

UV

(E = 9 x 103).

three

fitted

acid

C8H5N304:

be

was recrystallized

mp = 319-320°C

Calc.

perman-

was precipitated

acetanilide

When 1.7 g of 2-aminoisopthalic

a Rayonet

with

lit6

Anal.

--et al.

to 2, 6 dimethyl-

amine

C, 53,04;

H, 2.55;

with

anhydride

2-Aminoisopthalic

C, 46.56;

10 min.

H, 2.43;

of Blanchard

The unhydrolysed

mp = 250;

acid

by the method

Found:

were

g

less

3090m(C-H),

was subsequently

C8H7N04:

was recovered.

Samples for

which

for

N, 7.75;

acid.

30%) of azide

0.46

300 nm (E = 1.5 x 103),

of acetic

the pH to 1.

2-Azidoisopthalic azidoisopthalic

3455m(OH),

M phosphate)

3N NaOH yielded

Calc.

H, 4.02;

contained

C, 46.39;

of

yielding

mp = app 230°C

33540m(OH),

Addition

with

Anal.

C8H5N304:

was prepared

the acetanilide

by lowering

from water.

at 37'

the product

N, 19.90.

the pH to 4 and filtered.

recovered

Addition

and 220 nm (E = 2 x 104).

Hydrolysis

by lowering

and dried

that

for

UV (0.1

modifications6

afforded

Calc.

(KBr disk)

2-Aminoisopthalic with

was filtered

H, 2.52;

1600cm-1;

of N2 had subsided.

indicated

Anal.

C, 45.81;

mp = 250 (dec)

1715S(C=O),

which

by fluorescence

0.1% of the amine.

N, 20.28;

the bubbles

the azide

Analysis

than

until

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pyrex

with

filters

16 lamps

at room temperature having

maximum emission

at 310 nm. The Lineweaver-Burk 5 azidoderivatines

were

plot

shown

competitive

at the active

in Figure

inhibitors,

by binding

site.

inhibition

were

31 mM for

2-azidoisopthalic

In order

to reduce

the photoinactivation

that

and presumably

Under

activity

1 indicates

acid

these

conditions

and 12 mM for

both

the 2 and

affected

the cataly

the Ki's

for

5-azidoisopthalic

acid.

enzyme-inhibitor

complex

were

photolysed

of the enzyme, in

1131

test

tubes

surrounded

solutions by three

of

tic

BIOCHEMICAL

Vol. 52, No. 4, 1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

d

JO-

C

.c Z EEE I

I 2

I

3

x IO”

&!IK

FIGURE 1 1 Ex p erimental ; "' [a;G] conditions were 1.7 x IOe4M NADH, 5 mg/ml GDH, at pH = 7.3 plus the 0.05 M NH4C1, I.0 x 1O-4 M EDTA and 0.1 M phosphate following: (a) 0.05 M 5-azidoisopthalic acid, (b) 0.05 M 2-azidoisopthalic acid, (c) 0.025 M 2-azidoisopthalic acid and (d) none.

Pyrex

filters

with

conditions

only

photolysis

in

lamps having

~4% of the activity the absence

The photolysis buffer

for

monomeric

over products

this

appears

that

or six

resulting

Excitation

band

could little

of the enzyme was lost

were

from

against

to remove

the reaction

an emission from

Under

during

transfer

changes

these

a ten minute

of 0.1 M phosphate

unreacted

of nitrenes

azides with

band at 430 nm with

280-300

be attributed energy

dialysed

day period

enzyme showed

maximum at 350.

at 310 nm.

of the inhibitor.

solutions

a five

of the dialysed

but

a maximum intensity

nm showed

to the enzyme occurred

slight

itself

from

and any Analysis

water. excitation

emission and therefore

at 430 nm, it

the enzyme to the bound

inhibitor. Kinetic lost

measurements

on photolysis

dehydrogenase

if

Photolyses

with indeed were

indicated

each isomer modification also

carried

that

about

suggesting

50% of the activity that

of the active out

only site

about

4 of the glutamate

had occurred.

in an Aminco-Bowman

1132

had been

spectrophotofluorometer

BIOCHEMICAL

Vol. 52, No. 4, 1973

at 12'

for

because

36 hours

of the

with

lower

AND BIOPHYSICAL

the monochromotor

light

RESEARCH COMMUNICATIONS

at 350 nm but

the yield

was less

intensity.

DISUSSION These acid

are

experiments

have

competitive

nitrenes,

both

activity

shown

inhibitors

reacted

inhibitor

may lead

to identification

azides

with

it

is

bound

from

loss

to form

of catalytic

of the dialyzed

an affinity

label

from

to the enzyme catalytic

of the catalytic

isopthalic

When photolyzed

emission

means of generating after

prepared

the enzyme with

of a new fluorescence

This

competitive

the

of the GDH reaction.

irreversibly

and appearance

enzyme at 430 nm.

that

a

site

hopefully

site.

REFERENCES

1.

Pisezkiewicz, 2622

D.,

Landon,

M.,

and Smith,

E. L.,

--J.

J. Biol.

Chem.,

247,

Biol.

e.,

-,245

(1'970).

2.

Goldin,

3.

Baker,

4.

Drowles,

5.

Gevaert

B. R., B. R., J. R., Photo

and Frieden, Design

C.,

of Active-site

Accounts

Directed

--Chem. Res.

Production

N.V.

2139

Irreversible

(1972). Enzyme Inhibitors,

2, 155 (1971).

Belgium

Patent,

656511

(1965);

Chem. Abstr. --

63, p8584g. 6.

Blanchard, -Johns

K. C.,

-Hopkins

Dearborn,

Hospital,

91,

E. H., 330

Lasanga,

(1952).

1133

L. C.,

and Buhle,

E. L.,

Bull.

the