Oxidation of polyamines by pyrroloquinoline quinone (PQQ)

Tetrahedron Vol 4.5, No 13, pp 4235 to 4242, 1989 F’nnted I” Great Bntzun

ao40-4020/09 5300+ ccl 0 1989 Maxwell Pqamon Macmdlan pk

OXIDATION

OF POLYAMINES

PYRROLOQUINOLINE

G. Houen

1.

2.

1*

Department

of Molecular

81. Amager

Boulevard

Department

of

, c. Larsen*

General

Universitetsparken

Cell

and

State

Serum

Copenhagen

Organic

5. DK - 2200

(PQQ)

and L .-I. Larssonl

Biology.

80. DK - 2300

BY

QUINONE

Chemistry.

Copenhagen

Institute,

Building

S, Denmark. H.C.

Brsted

Institute.

N, Denmark.

(Received in UK 12 April 1989)

Pyrroloquinoline quinone (PQQ) was found to oxidize the polyamines spermine and spermidine in stoichiometric amounts with diaminopropane as the main product identified by reversed phase high performance liquid chromatography and mass spectroscopy of the reaction products. Putrescine was oxidized much slower than the polyamines and cetyl-trimethyl-ammonium bromide did not promote oxidation. A model for the PQQ oxidation of polyamines is suggested to account for these observations.

INTRODUCTION The

polyamines

necessary

concentration

are

oxldases

cell of

degradation. which

spermine

for

these

can

be

divided and

is

(SPD) and putrescine division

cell

regulated

of polyamines

catalyzing

oxidases)

and

molecules

The degradation

enzymes

monoamine

(SPM), spermidine

growth

the

into 2.

through

is catalyzed

oxidative two

(l-4).

degradation

classes:

PQQ-dependent

1.

also

group of the second class, has only recently

PQQ

(7-10).

catalytic investigated

Since

PQQ

oxidation

has

of

Cu-amine

been

amino

oxidases)

reported acids

to

(11)

the ability of PQQ to oxidize

biosynthesis

be

of

(5,6).

amines.

and

Amine (mainly

polyamine

The

organic

been identified

capable

and

and

by amine oxidases,

diamine

oxidases,

as

cellular

FAD-dependent

(mainly

prosthetic

classified

(PUT) are

The

amines

of

as

efficient

(12)

we

have

polyamines.

ABBREVIATIONS PQQ,

pyrroloquinoline

putrescine; fast

atom

phosphate;

quinone;

DAP, diaminopropane; bombardment CTAB,

-

SPM,

spermine;

SPD,

FAD, flavin adenine

mass

spectroscopy:

cetyltrimethylammoniumbromide;

resonance. 4235

spermidine;

dinucleotide;

TEAP, NMH,

PUT,

FAB-MS,

triethylammonium nuclear

magnetic

G. HOUEN et al.

4236

MATERIALS SPM'IHCl, USA).

SPD'3HC1,

(Buchs, Merck

Switzerland).

(Darmstadt,

Reactions HPLC

were

were

using

CTAB,

dansylated

ln 15 mln

and

detector,

analysed

dissolved

;n glycerol

was

done

on

as

(St. Louis,

were

from

and Na2HP04

buffer

pH 7.2.

by

FLUKA

were

from

Scotland). Samples

Brossat

et

for

al.

(13)

50% 50 mM TEAP pH 4, to 100% MeOH in 30 min

510 pumps,

a model

Data Module,

(B). HPLC was done 420-AC

fluorescence

a Z-module with a 8 mm x 10

System Controller.

were

dried,

extracted

into MeOH,

dried

again

and

for FAB-MS. a VG Masslab

VG

20-250

Quadropole

fitted with a VG FAB source and probe. The primary produced

PQQ

(Walkerburn,

described

to 100% MeOH

a Waters

and a Waters

the HPLC

NaH2P04

in phosphate

two model

a 710 B WISP,

from

NaCl,

from 50% MeOH,

system with

rnlnRP-8 column, Samples

NaOH,

from Sigma

and

MeOH was from Rathburn

at 37OC

gradient

were

diaminopropane,

(A) or from 60% MeOH

on a Waters

FAB-MS

H3P04,

Germany).

performed

a linear

and dansylchloride

triethylamine,

PUT'ZHCl,

AND METHODS

mass

spectrometer

beam of xenon atoms was

from an ion gun, Ion Tech. Ltd., operating

at 1.0 mA, 8 kV.

RESULTS In contrast had

to its effect

no measurable

the presence

ion PQQ exerted with

the

effect

effect

or absence

order

of

on the oxidation

of primary

polyamines

30

min

gradient

gradient

from

(Pig.

from

corresponding

in mmolar

oxidation In

SPM ) SPD ) PUT.

amines

(12) CTAB

concentrations

of polyamines,

dependent

both

in

concentrat-

of polyamines

contrast

to

had an inhibitory

50 to

oxidation

3) and PUT was

with these results

60%

100% MeOH

eluted

eluted

concentrations

to

100%

MeOH

(Table

two products of

SPM

oxidized

the

at a position

at a position

to DAP

for

its

effect

the

determination

corresponding

product

a small

of

eluted the

extent

precipitates,

at

peaks

compounds

a

was

from the manner

less effectively (Fig. 4).

it was found that only SPM and SPD reacted

PQQ at pH 4 forming orange and yellow react with PQQ to form a precipitate.

Using a

in a time dependent oxidized

of

to PUT/DAP

SPD.

to

identity

dansylated

were formed

to

HPLC

major

2). The

(Fig. 2). SPD was only

of PQQ. Using a 15

corresponding

by FAB-MS of the collected

(Table 2). These

in the PQQ

concentrations

of SPM at various

the major product

further confirmed HPLC

in pmolar

of SPM.

and the minor product

position

PQQ

(Table 1). However,

effectiveness:

on oxidation

linear

on mmolar of CTAB

amines

a time and concentration

Fig. 1 shows oxidation min

on primary

whereas

In line

rapidly with PUT did not

Oxidation ofpolyamines

TABLE 1. Effect ions

of PQQ on mM concentrations

of CTAB.

Samples

were

4231

of SPM at various

incubated

for

20 hours

concentratin phosphate

buffer pH 7.2 at 37OC.

SPM cont.

(-a

PQQ cont.

CTAB cont.

(mM)

(mM1

% Oxidation

1

0.001

1

0.001

2

0

1

0.001

20

0

0

0

0

0

10

0.01

10

0.01

2

0

10

0.01

20

0

1

1

0

80

1

1

2

46

1

1

20

20

TABLE 2. HPLC

and FAB-MS

analysis

of the products

from PQQ oxidation

of

SPM.

Peak nr.

Elution

timea)

(min.)

Massb)

1 (major)

14.66

0.06

540

Compound c) DAP

2 (minor)

26.98

0.02

670

SPD

STD A

14.69

0.05

540

DAP

STD B

15.73

0.00

554

PUT

670

SPD

STD C

27.10

0.02

STD D

29.53

0.03

a)

Gradlent

B

b)

Mass of the dansylated

c)

Name of the un-dansylated

compound compound

SPM

G. HOUJZN et al.

4238

Fig. 1 Effect

of PQQ concentration on oxidation of SPM. Samples were

incubated in phosphate buffer

pH

7.2 at

37°C

for 20 hours.

Polyamine concentrations were determined by HPLC.

Samples were incubated in phosphate buffer pH 7.2. PQQ concentration was 1 mM.

Fig. 2 Time

course

for

the

oxidation of

SPM by

PQQ.

Oxidation ofpolyamines

Fig. 3 Time

course

for

the

incubated in phosphate

oxidation

of

SPD

4239

by

PQQ.

Samples

were

Samples

were

buffer pH 7.2 with 1 mM PQQ.

I t 10

20

30

!50

40

Time San1

Fig. 4 Tlae

course

incubated

for

the

in phosphate

oxidation

of

PUT

by

PQQ.

buffer pH 7.2 with 1 mM PQQ.

4240

G. Horn

et al.

DI!XWSSION The inability by

assuming

of CTAB to promote a repulsion

by an inability In

any

case,

oxidation oxidized

of CTAB PQQ

of

is

an At

with

order

is the identification

PQQ

of

oxidation

SPD is PUT by

a

catalyzed

secondary

oxidation

aminoaldehyde, released

and

concomitant be assumed forces

case

al., enzyme

of

by

500 MHz

unpublished

1 H-NMH

5).

of PUT

product

from the from

the

of SPM and

This may be explained preferentially

group with

In analogy

adduct

PQQ

however,

oxidation

carbonyl

may

the more

with

then

the PQQ

split

to

an

and PQQ bound DAP. DAP may then be action

of

In the enzyme

02

and

catalyzed

H20

with

reaction

the

it can

imposed by the enzyme on the reaction, to take place

on the other

studies

of the enzyme of these

catalyzed

This

oxidation

the

found

to be

(G. Houen role

et

of

the

of polyamines

can

of the site of cleavage

of DAP. 2. Efficient

of the energy

was

reaction

results,

1. Steric control

instead

side of the

of PUT from SPD and SPD from SPM with

In light

amine oxidase

3. Lowering

of the reaction.

the

the

SPM ) SPD > PUT.

polyamines

3-amino-propionaldehyde.

to be threefold:

formation

the catalyst.

of

results).

in the plasma

assure

of H202.

the C-N bond

formation

the

(Fig.

the

N-atom with the formation

be considered to

by

that steric constraints

concomitant the

atom acids,

main

results).

where

cyclizes,

regenerated

generation

cleavage

secondary

amino

easily

the

catalysed

on the cofactor

nitrogen

of

which PQQ

since oxidase

mechanism,

attack

for

of DAP as the main product

SPD, amine

effectiveness:

or

of polyamines.

catalyst

concentrations,

of

et al., unpublished

reaction

a nucleophilic

nucleophilic

and

plasma

(G. Houen

assuming

makes

SPM

bovine

autorecycling

equimolar

the

may be explained

and the polyamines

in the presence

effective

Also surprising

PQQ-dependent

of polyamines

the CTAB micelles

to form micelles

not

polyamines.

polyamines

oxidation

between

of activation,

regeneration

of

i.e. "catalysis",

4241

Oxidationofpolyamines

HOOC

“,o,+“~~~

noocd$oo~n~cn*cn~~~%C-

coon HOOC

HOOC

M

’ ‘N

HOOC

\

7

1;

‘I HOOC ‘N r-Q!

OH

HwHFHF”~I;

“flCH,C&C”fN\”

0 OH CW”C”FW’“Y

OHCCH&H,CH,NH,

Fig. 5 Possible

reaction mechanism

for the PQQ oxidation

of polyamines.

4242

G. HOUEN et al.

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ACKNOWLEDGEMENTS The

Danish

financial

Cancer

Society

and

the Lundbeck

support. Vibeke Nielsen

is thanked

Foundation

are thanked

for

for typing the manuscript.