Tropylium tetrafluoroborate, a novel substrate for aldehyde oxidase

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

October 30, 1986

Pages 609-615

TROPYLIUM TETRAFLUOROBORATE, R. Bruce

A NOVEL SUBSTRATE FOR ALDEHYDE OXIDASE

Bank@

and Stewart

D. Barnett

Department of Chemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412 Received

September

15,

1986

SUMMARY. The stabilized carbonium ion salt, tropylium tetrafluoroborate, was oxidized to tropone (cycloheptatrienone) by rabbit liver aldehyde oxidase related molybdenum hydroxylase, xanthine oxidase. but not by the closely The tropylium cation is an aromatic hydrocarbon which lacks the aldehyde, or iminium functional groups present in other substrates of aldehyde imine, oxidase. The unique structural features of the tropylium ion should make 'J 1986 Academic Press, it a useful tool for mechanistic studies of aldehyde oxidase. Inc.

The molybdenum oxidase

(E.C.1.2.3.2),

containing have in

mally

of xanthine

All contain

of

the

enzymes dized

(4). functional

nitrogen

Oxidation

It

these has group

or oxygen is

oxidase

reported

of

the

which

(2).

of

undergoes

*To whom correspondence

should

aldehyde

occurs

proposed

by the

oxidase

in conjugation

substrates

achieved

of

compounds

These

xenobiotics

hydroxylases into

such

enzymes

are the

reactions

for-

products are

and of pyridinium

the salts

(1).

carbon

serves

of

of several

introduced

Examples

by xanthine

substrates

been

(1,2).

molybdenum

oxygens

and xanthine

number

groups

and detoxication

in

acid

a large

functional

by the

to uric

(E.C.1.2.3.1)

of

catalyzed

water

an electrophilic

oxidation

iminium

from

by aldehyde

oxidase

oxidation

the metabolism

processes derived

the

or

Oxidations

ultimately

to pyridones

the

in

oxo-transfer

oxidation

The

imine

been implicated (3).

aldehyde

catalyze

aldehyde,

mammals

are

hydroxylases,

that attack

the

and

be addressed.

oxidase

a nitrogen

or oxygen

the

molybdenum

centers

electrophilic

to the

transfer

xanthine

with

carbon

by an active-site

as a ligand coupled

at

oxidase

of

nucleophile,

catalytic two

of

molybdenum

electrons

and

atom. of

the

the oxiwhile

center. a proton

Vol. 140, No. 2, 1986

Figure

with

BIOCHEMICAL

AND BIOPHYSICAL

I

II

III

1. Structures of the tropylium cation (I),

reduction

of Mo(V1) to Mo(IV);

by oxygen or other electron

RESEARCH COMMUNICATIONS

tropyl alcohol (II),

the enzyme is

and tropone (III).

subsequently reoxidized

acceptors (5,6).

Although aldehyde oxidase and xanthine oxidase are similar and catalytic

mechanism, they differ

between the enzymes are especially

in substrate specificity

(7).

in structure Differences

apparent in the case of charged substrates.

For example, aldehyde oxidase catalyzes

the oxidation

quinolinium and other iminium ions at neutral

of numerous pyridinium,

pH, while xanthine oxidase acts

on these cations only at higher pH (8,9). The tropylium sesses certain oxidase.

ion (Figure

l,I),

although lacking

molecular features in commonwith charged substrates of aldehyde

Like pyridinium

salts,

it

few carbonium

lar

(10).

was investigated

its

tropyl

this

Because its

salts

ion is one of the in aqueous solution;

with the hydrolysis structure

product,

tropyl

and charge are very simi-

and other iminium ions,

that

communication we report form) is oxidized

tropylium

to tropone (III)

but not by bovine milk xanthine

this report provides the first as well as the first oxidation

electronic

an aromatic ring

the tropylium

ion

as a substrate for the molybdenumhydroxylases.

alcohol

hyde oxidase,

The tropylium

ions which is stable enough for existence

to those of pyridinium

In

(10).

pH the cation is in equilibrium

alcohol (II)

contains

is cationic,

system, and reacts with nncleophiles

at neutral

an iminium group, pos-

by rabbit

oxidase.

example of a tropylium

case of acationic

tetrafluoroborate

(or

hepatic alde-

To our knowledge,

cation biotransformation,

hydrocarbon acting

as a substrate

for

by aldehyde oxidase. MATERIALSANDMETHODS

chloride, menadione Reagents. Xanthine oxidase (Grade I), l-methylnicotinamide acid ferric-sodium salt were purchased from and ethylenediamine tetraacetic 610

Vol. 140, No. 2, 1986

BIOCHEMICAL

Sigma; potassium monohydrogen from Fisher; Triton X-100 ferricyanide from Mallinkrodt. tropone were gifts from Dr. Greensboro). The tropylium trile, and tropone was purified of these compounds (uv, nmr, (11-13).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and dihydrogen phosphates and potassium cyanide from U.S. Biochemical Corporation and potassium Samples of tropylium tetrafluoroborate and (University of North CarolinaJames C. Barborak tetrafluoroborate was recrystallized from acetoniby chromatography on silica gel. The spectra ir) were identical with those previously reported

Enzyme assays. Aldehyde oxidase was partially purified from rabbit livers (Pel-Freez Biologicals) by ammonium sulfate fractionation of the heat-treated homogenate (14). Control aldehyde oxidase activity was measured by the method of Rajagopalan and Handler using l-methylnicotinamide chloride as substrate and potassium ferricyanide as electron acceptor (15). Protein concentrations were assayed by the Lowry method (16). Enzymatic oxidation of tropylium tetrafluoroborate was determined spectrophotometrically by measuring the rate of disappearance of the 420. nm band of ferricyanide. Sample cuvettes contained potassium phosphate (pH 7.8) or sodium carbonate (pHU.6) buffer (100 umole), EDTA (0.2 umole), aldehyde oxidase preparation (3 mg), potassium ferricyanide (3.0 umole), tropylium tetrafluoroborate (0.15-75.0 umole), and deionized water to give 3.0 ml final volume. Reference cuvettes contained deionized water instead of substrate. Reactions were initiated by addition of substrate after preincubation of cuvettes at 37" for five minutes. Inhibitors, when present, were included during the preincubations. Initial rates of ferricyanide reduction were determined over the first two minutes of reaction; activities were calculated using the extinction coefficient of ferricyanide Fixed wavelength measurements and repetitive (E 220 = 1040 M-l cm- ') (7). spectral scans were recorded on a Hitachi 100-80 model spectrophotometer equipped with thermostatted cell holders and program mode for enzyme kinetic analysis. Calculation of kinetic constants. Michaelis-Menten kinetic parameters were calculated by non-linear regression of substrate concentration and initial velocity data using the computer program of Duggleby (17); initial estimates of the parameters were determined from Lineweaver-Burk plots. (18) HPLC Analysis. Ten flasks, each containing potassium phosphate buffer (100 umole; pH 7.8), aldehyde oxidase (6 mg), tropylium tetraEDTA (0.2 umole), fluoroborate (1.5 nmole) and deionized water to give 3.0 ml final volume, were incubated at 37" overnight and then combined. Controls containing heatdenatured aldehyde oxidase were incubated under identical condition. The samples and controls were extracted with ethyl acetate and the extracts dried Analyses were performed using a Perkin-Elmer over anhydrous sodium sulfate. C-18 reverse-phase Tridet system equipped with SSI Model 300 pump, an Alltech column and uv detector (256 nm). Methanol-water (70:30) was used as the mobile phase. RESULTS Tropylium hyde of

tetrafluoroborate

oxidase the

pylium

as measured

oxidation

served

by the

was greatly

oxidation

occurred

cess

was very

slow

tron

acceptors

for

mate

(19),

were

in

compared aldehyde

also

reduced

as a substrate

reduction

of

diminished the

at

ferricyanide pH 10.6.

absence

of enzyme,

that

the

with oxidase, when

of

for

enzymatic

rabbit (Table

A small but

the

rate

611

to

incubations

the

rate

amount

of

tro-

of this Other (14)

of

the

alde-

1);

reaction.

such as dichloroindophenol added

liver

proelec-

and chro-

tropylium

ion

Vol. 140, No. 2, 1986

Table

BIOCHEMICAL

1. Oxidation

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of Tropylium Tetrafluoroborate by Molybdenum Hydroxylases: Effect of Enzyme and pH Oxidase Activity

Enzgme

(ml

substrate

oxidized/min/mg

pH 7.8

Aldehyde Oxidase Xanthine Oxidase

Protein)

pH 10.6

0.155 + 0.014 0.001 * 0.001

0.037 + 0.006 0.002

f 0.001

Ferricyanide assays were performed as described in Materials and Methods. Incubations contained l.OmM tropylium tetrafluoroborate. Assays with xanthine oxidase were carried out in the presence of 3.0 mg of enzyme. Reported activities are corrected for nonenzymatic oxidation and represent the mean ? S.D. of three experiments. Control aldehyde oxidase activity was 0.063 umole 1-methylnicotinamide chloride oxidized/min/mg protein; xanthine oxidase activity was 0.074 umole xanthine oxidized/min/mg protein. The rates of nonenzymatic tropylium oxidation were approximately 0.004 (pH 7.8) and 0.003 (pH 10.6) umole/min/mg protein.

with

the

enzyme.

for

xanthine The

oxidase rate in

compounds

which

the

the

by aldehyde

partially

purified

inhibit the

oxidase,

rather

than

inhibition incubated

Effect

2.

occurred with

when high aldehyde

oxidase

or

(Table of

by other

the

X-100,

(20).

tropylium

These ion

was

in

the

present

concentrations oxidase;

of tropylium

this

inhibition

Oxidation

% Inhibition 0

Menadione Triton

was

Triton 2)

enzymes

of Aldehyde Oxidase Inhibitors on Enzymatic Tropylium Tetrafluoroborate

--(3.3 X-100

Potassium

increase

a substrate

tetrafluoroborate

menadione,

oxidation

Compound

Oxidation

tropylium

cyanide,

observed

as

pH values.

of

aldehyde

inactive

preparations.

were

Table

that

and basic

potassium

to

was

oxidation

of

known

catalyzed

fluoroborate

enzymatic

suggest

Substrate

neutral

presence are

strongly

tetrafluoroborate

at both

of

decreased

data

Tropylium

rates

uM) (8

Cyanide

99

x lo-' (0.2

X)

76

mM)

46

were

determined spectrophotometrically by measuring the at 310nm for tropone formation. Assay mixtures phosphate buffer (33mM; pH 7.8), tropylium tetrafluoroborate oxidase (lmg/ml). Values represent the mean of two control aldehyde oxidase activity was 0.063 nmole chloride oxidized/min/mg protein.

in absorption

contained potassium (ImM), and aldehyde experiments. The 1-methylnicotinamide

612

tetrais

illus-

of

Vol.

140,

No. 2, 1986

0

AND

BIOPHYSICAL

03

,’

I

-5

2

BIOCHEMICAL

0

5

11s

IO

15

lo

RESEARCH

0 I 240

COMMUNICATIONS

300 350 WAVELENGTH (nm)

400

Figure 2. Lineweaver-Burk plot for the oxidation of tropylium tetrafluoroborate by rabbit liver aldehyde oxidase. Assays were performed as described in Materials and Methods. Calculations were carried out with data from three experiments, each in duplicate. Initial velocities were expressed in nmoles substrate oxidized/min/mg protein and substrate concentrations were in millimolar. KP, = 1.15 + 0.09 mM; vmax = 0.33 + 0.02 umoles/min/mg protein (mean + S.D.). Figure 3. Time-dependent spectroscopic changes during oxidation of tropylium tetrafluoroborate by rabbit liver aldehyde oxidase. The sample cuvettes (1 cm pathlength) contained potassium phosphate buffer (100 umole; pH 7.8), EDTA (.2 nmole), tropylium tetrafluoroborate (1.5 umole), aldehyde oxidase (3 mg), and deionized water to give 3.0 ml final volume; the reference cuvette contained all components except tropylium tetrafluoroborate. Spectra were recorded from 450-240 nm at 3-minute intervals for 30 minutes (37").

trated

in

velocity

the

double

data

(Figure

oxidase-catalyzed portion

ion

plot,

order

to

oxidation,

incubation was

band

at

point

was

prevented run is (Amax

obtain

ion

nm and

observed when

with

273

substrate

(2).

The apparent

nm) cannot

f 0.09mM

at

growth 273

nm.

was of

be directly

the

a new

These

initial

is

in

aldehyde

product

alcohol; observed 613

from

formed scans

As shown

band

of at

the

the

during

were

linear

in

Figure

the

absorption

were The

incubation. by

at

pH,

neutral

during

3, the oxida-

initial

changes

absorption

tropylium

recorded

311 nm; a clean

spectroscopic during

common

t S.D.).

disappearance

present

tropyl

(mean

oxidase.

of

and

Km, calculated

spectroscopic

aldehyde

concentration

inhibition

on

by progressive the

substrate

Such

information

menadione

characteristic

of

uv-visible

accompanied 256

plot

was 1.15

repetitive of the

tion

2).

reactions

of the In

reciprocal

the since

isosbestic completely band

tropyl the

at

256

cation alcohol

Vol. 140, No. 2, 1986

is

the

at

311

BIOCHEMICAL

predominant nm is

nm) as the

consistent

oxidation

when organic sis.

component

only

of extracts

denatured

of

the

from

was

were

time in

Xmax = 312

product

was confirmed

subjected

with

to HPLC analy-

active

as a reference

extracts

absorption

(reported

of the

incubations

present

The observed

tropone

incubations

same retention

tropone

of

The identity

scale

RESEARCH COMMUNICATIONS

(21).

formation

(12).

of large

peak with

a trace

equilibrium

the

product

The chromatogram

ed a large

of the

with

extracts

AND BIOPHYSICAL

from

enzyme containsample

of tropone;

incubations

with

heat-

enzyme. DISCUSSION

Tropylium We,

imine

hyde

oxidase.

is

tetrafluoroborate or

to

to

in

in

the

oxidize

specificity

of

a carbonium groups

presented

tropone

oxidase

substrate

functional

Data

oxidized

aldehyde

iminium

is

the

present

this

of

tropylium

latter

salt

in

report

presence

the

ion

other

establish

aldehyde salt

enzyme,

which

lacks

substrates

of alde-

that

compound

this

oxidase.

further

the alde-

The

ability

underscores

particularly

toward

of

the

broad

cationic

com-

pounds. Aqueous mixture

of

latter it

solutions the

predominates is

for

the

aldehyde

the

the

substrates

of

covalent ions to often

be

the

equilibria

proposed

several

substrates

actual

Oxidation

the the

alcohol

consistent

decrease of

similar

aldehyde

true

cationic

the

as aldehydes

for

the

with

such

salts,

is

in the rate

that

iminium

neutral

do not establish

which

increasing

are

at

reduction

pH should

which

of an equilibrium

(II);

Our data

observed

the

consist product

(21).

tropyl

more

oxidase,

For

inhibitory

or

while

aldehyde

water.

its

= 1.8 x lo-')

increasing

cation

hydration

with

hydrolysis

and

pH is

since

of

(I)

However,

higher

substrate,

tetrafluoroborate

eq cation

oxidase. at

tration

(K

tropyl

oxidation

isms

cation

of tropylium

the

the and to

the

equilibrium

free

oxidase;

the

acting

as

concenOther

salts,

undergo

reaction cations

substrate

alcohol.

iminium

the

whether

of tropylium

form

tropyl

pH the

of

tropylium

have been

hydrated

forms

shown are

(22,23). of for

the the

tropylium action

cation of

can be rationalized

aldehyde 614

oxidase

(5,6).

in

terms For

of mechan-

example,

the

Vol.

140,

No. 2. 1986

tropylium hydride in

ion

could

transfer

analogy

with

those

act

ion

of

of aldehyde

oxidase.

or

as substrate

pylium

cation

of aldehyde

for should

serve

as the

donor

oxidase-catalyzed

is

enzyme.

make it

ion ligand

not

The unique

a useful

probe

and

subsequent

hydrolytic

oxidation;

the

steps

H-electrons

to molybdenum. to tropone

indicate a strict

COMMUNICATIONS

enzyme with

transfer)

iminium

results

RESEARCH

by the

tetrafluoroborate

Our group

the

attack

for

tropylium

iminium

BIOPHYSICAL

proton-electron

proposed

could

AND

nucleophilic

coupled

The oxidation

imine,

We,

undergo (or

of the troplium

tion

BIOCHEMICAL

that

the

presence

requirement structural for

the

is

for features

study

of

a novel

reac-

of an aldea molecule of the the

to tro-

mechanism

oxidations. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Rajagopalan, K.V. (1981) Enzymatic Basis of Detoxication, Jakoby, W.J., Ed., pp. 295-309, Academic Press, New York. Bray, R.C. (1975) Enzymes 12, 299-419. Beedham, C. (1985) Drug Metabolism Reviews 16, 119-156. Coughlan, M.P. (1980) Molybdenum and Molybdenum-Containing Enzymes, pp. 138-140, Pergamon Press, New York. Olson, J.S., Ballou, D.P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382. Stiefel, E.I. (1973) Proc. Nat. Acad. Sci. 70, 988-992. Krenitsky, T.A., Neil, S.M., Elion, G.B., and Hitchings, G.H. (1972) Arch. Biochem. Biophys. 150, 585-596. Greenlee, L., and Handler, P. (1964) J. Biol. Chem. 239, 1090-1095. Bunting, J.W., Laderoute, K.R., and Norris, D.J. (1980) Can. J. Biochem. 58, 394-398. Harmon, K. M. (1973) Carbonium Ions, Olah, G.A. and Schleyer, P. von R ., Eds., pp. 1579-1641, Wiley-Interscience, New York. Harmon, K.M., Cummings, F.E., Davis, D.A., and Diestler, D.J. (1962) J. Am. Chem. Sot. 84, 120-121. Dauben, H.J., and Ringold, H.J. (1951) J. Am. Chem. Sot. 73, 876-877. Harmon, K.M., Harmon, A.B., and Thompson, B.C. (1967) J. Am. Chem. Sot. 89, 5309-5311. Rajagopalan, K.V., Fridovich, I., and Handler, P. (1962) J. Biol. Chem. 237, 22-29. P. (1966) Methods in Enzymol. 9,364-368. Rajagopalan, K.V., and Handler, Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Duggleby, R.G. (1981) Anal. Biochem. 110, 9-18. Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Sot. 56, 658-666. Banks, R.B., and Cooke, R.T. (1986) Biochem. Biophys. Res. Commun. 137, 8-14. Rajagopalan, K.V., and Handler, P. (1964) J. Biol. Chem. 239, 2022-2026. Doering, W. Von E., and Knox, L.H. (1954) J. Am. Chem. Sot. 76, 32033206. Brandige, S., and Lindblom, L. (1979) Biochem. Biophys. Res. Commun. 91, 991-996. Thomas, H.G., and Reunitz, P.C. (1984) J. Heterocyl. Chem. 21, 1057-1062.

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