Irreversible inactivation of pig brain γ-aminobutyric acid-α-ketoglutarate transaminase by 4-amino-5-halopentanoic acids

Vol.

95. No.

July

16, 1980

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

250-255

Pages

IRREVERSIBLE INACTIVATION OF PIG BRAIN y-AMINOBUTYRIC c+KETOGLUTARATE TRANSAMINASE BY 4-AMINO-5-HALOPENTANOIC ACIDS

ACID-

Richard B. Silverman and Mark A. Levy Department of Chemistry, Northwestern University Evanston, Illinois 60201 Received

May

29,198O

SUMMARY y-Aminobutyric acid-ar-ketoglutarate transaminase from pig brain is irreversibly inactivated by 4-amino-5-halopentanoic acids. Protection from inactivation by the natural substrates, the pH dependence of inactivation and the incorporation of 1.7 moles of radioactive inhibitor per mole of enzyme from (S)-[UJ*C]-4-amino-5-chloropentanoic acid suggest a covalent adduct at the active site of the enzyme. A mechanism-based inactivation. is proposed. INTRODUCTION It is well established Its concentration converts

in the brain

glutamate

of GABA to succinic a coenzyme vulsions

to GABA,

may result

(2).

(1).

diseases,

been to block the degradation used epilepsy reversibly

(4).

drug,

sodium

Several

neurotransmitter

by two enzymes,

and GABA-T,

GAD, which

which catalyzes

Both of these enzymes

When brain

levels

(1).

the degradation require

of GABA diminish,

PLP

(3), a chemotherapeutic e.g.,

epilepsy

and Huntington’s

of GABA by inhibiting valproate,

compounds,

approach

GABA-T

barrier

to the treatchorea, (1).

has been shown to inhibit 4-aminohex-5-ynoic

as

con-

Since GABA does not cross the blood-brain

circumstances

ment of convulsive

is regulated

semialdehyde.

for activity

under normal

that GABA is an inhibitory

has

The widely GABA-T

acid (6), 4-amino-

Abbreviations: GABA: y-aminobutyric acid; GABA-T : Y-aminobutyric acid -a-ketoglutarate transaminase, E.C. 2.6.1.19; GAD: glutamate decarboxylase, E .C. 4.1.1.14; PLP : pyridoxal phosphate; a-KG: a-ketoglutaric acid; SSADH : succinic semialdehyde - NADP : oxidoreductase, E .C D 1.2.1.16; NADP : nicotinamide adenosine triphosphate; Pyr in Scheme 1 represents the substituted pyridine nucleus in PLP . 0006-291X/80/130250-06$01.00/0 Copyright IG 1980 by Academic F’ress, Inc. A II righrs oJ reproducilon in an.vJorm rererved.

250

Vol.

95. No.

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

hex-5-enoic

acid (7), gabaculine

(8), isogabaculine

sulfate

(lo),

have been proven

to be mechanism-based

vators

(5) of GABA-T.

GABA-T

(ll),

pentanoic results

Based on the accepted

we have synthesized

acids (9 as potential of our inhibition

NH+

L

anticonvulsant

studies with purified

irreversible

inacti-

of action of

of 5-substituted-4-amino-

agents and report pig brain la,

X

here the

GABA-T. X=F

,b, x = Cl coo-

2, X=Br

3

MATERIALS

(9), and ethanolamine-o-

mechanism

(12) a series

COMMUNICATIONS

cl, X=OH

AND METHODS

Pig brain GABA-T was purified by the method of John and Fowler for the rabbit brain enzyme (13). The enzyme showed one band on SDS gel electrophoresis and had a specific activity of 7.5 units per mg protein. One unit is defined as the amount of enzyme which catalyzes the transamination of 1 clmole of GABA per min at 25’ C . GABA-T activity was determined using a modification of the method of Scott and Jakoby (11). The final concentrations in the assay solution were 10 mM GABA, 5 mM a-KG, 1 mM NADP, 5 mM .R-mercaptoethanol, and excess SSADH in 50 mM potassium pyrophosphate buffer, pH 8.5. Enzyme activity was determined by observing the change in absorbance at 340 nm at 25’ C after several minutes of incubation. SSADH was prepared from GABASE (Sigma Chemical Co. or Boehringer Mannheim) by inactivating the GABA-T with (S)-4-amino5-fluoropentanoic acid followed by exhaustive dialysis at 4’ C against 37.5 mM potassium phosphate, pH 7.2 containing 12.5% glycerol. This preparation could be stored for several months at -80’ C with no loss of SSADH activity and no gain of GABA-T activity. (S)-[U-14C]-4-Amino-5chloropentanoic acid (specific activity 1.02 mCifhnmo1) was prepared in an overall 32% yield from [U-14C]-L-glutamic acid (Research Products International) diluted with 0.25 mmol of unlabeled L -glutamic acid by carrying out the synthesis of & as previously reported (12). The purity was determined to be greater than 99% by TLC (cellulose, n-butanol-H,O-acetic acid, 12:5:3 and n-butanol-pyridine-water, 1:l:l) and by high voltage electrophoresis (pH 1.9). Radioactivity was determined on a Beckman LS-3100 scintillation counter using 3a 70B scintillation fluid (Research Products International) with [U-14C] toluene (New England Nuclear) as an internal standard. All other materials and methods have been reported previously (12) or are commercially available.

251

Vol.

95. No.

1. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

50

I.01 5

IO

I5

20

TIME

Figure 1.

Inactivation

of GABA-T

(min.

25

30

35

)

by (S)-4-Amino-5-fluoropentanoic

acid.

Pig brain GABA -T (0.13 8 units) was incubated at 25’ C in 0.4 ml of 50 mM buffer containing 5 mM mercaptoethanol and different concentrations of inhibitor and substrates. The incubation was stopped by diluting a 40 d aliquot of this mixture to 1 ml in a U. V. cuvette with buffer containing all of the necessary reagents for the assay. The inhibitor concentrations and the substrates depicted are: A , control; X, 0.100 mM inhibitor, pH 7.5; A , 0.200 mM inhibitor, pH 8.5; 0 , 0.100 mM inhibitor, pH 8.5; 0.100 mM inhibitor, pH 8.5, 0 , plus 1.0 mM cr-KG and Cl , plus 1.0 mM (u-KG and 1 .O mM GABA.

RJ3SULTS

AND DISCUSSION

The 4-amino-5-halopentanoic irreversible with

inactivators

of pig brain

b or a led to a pseudo

activity (PI-I 8.5,

(Figure

1).

(A3

GABA-T.

first-order

The KI and kc&

25’ C) and for*

It is noteworthy

acids

k, cJ were

Incubation

time-dependent (6) for&

252

of GABA-T loss

of enzyme

are 395 @I and 0.50 min -1

are 13.1 mM and 0.55 min-‘,

that the I$ for & is 12 times

found to be

lower

respectively.

than the KM (4.8 mM)

Vol.

95. No.

1, 1980

BIOCHEMICAL

for GABA (14).

Incubation

loss of enzyme vation

activity

gradually

AND

BIOPHYSICAL

of GABA-T

with & showed a time-dependent

In a separate

with time.

that preincubation

of & in pH 8.5 buffer

minutes

a solution

High voltage

amino-5-hydroxypentanoic

GABA-T

inhibitor

by 2 and l& is slower for the enzyme activity

(14).

dependent.

Since the rate of inactivation

strate,

The enzyme was protected

a-KG,

was dependent

and by GABA with a-KB on substrate

site-directed.

ethanol,

suggesting

that a reactive

Treatment followed

by exhaustive

ation of the labeled radioactivity inactivator

may be enzymeby its second sub-

The degree of protection

was unaffected

dialysis

does not escape from the active Exhaustive

enzyme

led to incorporation

This is consistent

two moles

of PLP

dialysis

of the inacti-

any enzyme activity.

thus confirming

to the protein. 253

acid

of 1.7 moles

of inacti-

with the fact that the enzyme

per mole of enzyme

in 8 M urea resulted

from the enzyme,

is

by B-mercapto-

with (S)-[U-14C]-4-amino-5-chloropentanoic

vator per mole of enzyme. containing

with the

that the inactivation

at pH 5.4 to pH 8.5 did not regenerate

of GABA-T

is a dimer

species

to effect inactivation.

1).

implying

The rate of inactivation

site and then return vated enzyme

concentration,

l), the pH optimum

inactivation

(Figure

of

of GABA-T

corresponds

of inactivation from

4-

is slower than the

The rate of inactivation

the mechanism

the enzyme.

the rate of inactivation

at pH ‘7.5 than at pH 8.5 (Figure

of the enzyme,

for 20

which we have found to be

of the experiment

hydrolysis.

we found

showed that it may contain

Presumably,

by g under the conditions

experiment

did not inactivate

a compound

of GABA-T.

rate of its non-enzymatic

active

of this solution acid (l&),

the rate of inacti-

in the absence of enzyme

whose components

electrophoresis

a reversible

COMMUNICATIONS

which was not pseudo first-order;

diminished

produced

RESEARCH

(14).

Denatur-

in little

or no release

covalent

attachment

of

of the

Vol.

95, No.

1, 1980

BIOCHEMICAL

Scheme 1. Proposed Mechanism 5 -halopentanoic Acids. These results inactivators

are currently

BIOPHYSICAL

of Inactivation

RESEARCH

. We believe

inactivators investigating

acids are irreversible

that these compounds

of this enzyme this proposed

COMMUNICATIONS

of GABA-T by 4-Amino-

show that 4-amino-5-halopentanoic

of GABA-T

based (suicide)

AND

as described mechanism

are mechanism-

in Scheme 1, and

of inactivation.

ACKNOWLEDGMENT The authors Institutes

are grateful

of Health

for support

of this research

by the National

(Grant NS 15703).

REFERENCES 1.

Roberts, Nervous

E., Chase, T. N., and Tower, System Function, Raven Press,

D. B. (1976) GABA in N. Y.

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Beeler, 371.

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Meldrnn, B. S. and Horton, R. W. in Harris, (Edit.) (1974) Epilepsy, Churchill Livingstone,

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Metcalf, B. W. and Jung, 539 -545.

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