A kinetic study of the effects of hydrogen peroxide and pH on ascorbate oxidase

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November 29,1978

Pages

A KINETIC

655-661

STUDY OF THE EFFECTS OF HYDROGEN PEROXIDE AND pH ON ASCORBATE OXIDASE

Ronald Department Received

E. Strothkamp

of Chemistry,

September

and Charles

Columbia

R. Dawson

University,

New York,

N.Y.

lDO27

27,1978 SUMMARY

Addition of hydrogen peroxide to ascorbate oxidase results in formation of a complex which has been analyzed kinetically. Since reduction of molecular oxygen to water by this enzyme occurs in more than one step, the peroxide complex may be a mimic for a catalytic intermediate. The properties of the complex are similar to those reported for a peroxide complex with laccase. Changes in the activity of ascorbate oxidase as a function of pH indicate the presence of a group in the enzyme with an apparent pK of 7.8 which must be protonated in order for the enzyme to function. The ascorbate Km is known to be insensitive to pH in this region and the present work indlcates the same to be true for the oxygen It would appear that the rate determining step in the catalytic mechanism % nvolves protonation of an intermediate. INTRODUCTION Ascorbate

oxidase

and ceruloplasmin dases, view

all

oxidases (2,3).

comprise

of which

on these occurs

via

electrons,

reduction

a class

possess

enzymes

The reduced

of four

(EC 1.10.3.3,

has recently

possibly

via

oxygen

Evidence

lactase

and ceruloplasmin

(5,6).

catalytic

results intermediate

(7,8).

of the effect

of hydrogen

of the

oxidases

"blue"

function

oxi-

of the

re"blue"

from

a suitable

substrate

oxygen

to water

with

addition

transfers

addition

Based on these on ascorbate in a similar

(4).

Assuming

which

results, oxidase manner,

for

peroxide

to rest-

may be related it

was felt

to a

that

was in order.

their

reaction

the

inter-

has been observed

of hydrogen

of a complex

the

a peroxide-like

such a species

Also,

peroxide

The reduction

in two steps,

for

in the formation

(1).

copper

and a comprehensive

two-electron

occurs

be formed.

known as "blue"

lactase,

transfers

reduce

two,

must

lactase

appeared

enzyme can then

oxidoreductase),

properties,

one-electron

mediate

ing

of enzymes

many similar

several

of molecular

1-ascorbate:02

a study If all

with

OOOS-291X/78/0852-0655SOl.O0/0 655

Copyright All rights

0 1978

by Academic

of reproduction

in any

Press, Inc. form

reserved.

BIOCHEMICAL

Vol. 85, No. 2, 1978

hydrogen the

peroxide

pH dependence

information of these

should

be comparable.

of the

activity

concerning studies

AND BIOPHYSICAL

will

It

was also

of ascorbate

the mechanism be presented

RESEARCH COMMUNICATIONS

felt

oxidase

of oxygen

that might

reduction.

a close reveal

look

at

additional

The results

of both

here.

MATERIALS

AND METHODS

Ascorbate oxidase was obtained from green zucchini squash (Cucurbita pepomedullosa) as previously described (9), and was homogeneous by polyacrylamide gel electrophoresis at pH 9.5 performed according to the procedure of Davis (10). All reagents were of the highest purity available and all aqueous solutions were prepared with deionized water just prior to use. Protein concentration was determined according to the Lowry procedure (ll), using bovine serum albtimin as standard. Copper determinations were performed according to Stark and Dawson (12). Enzymatic activity was determined at 25' C in a 1.5 ml reaction volume using a Clark oxygen electrode on a Gilson Model KM Oxygraph, under concentration and buffer conditions previously described (13). The formation of a hydrogen peroxide complex with ascorbate oxidase was followed by observing absorbance changes with a Cary I7 recording spechrophotometer, utilizing a thermostated cell holder maintained at 25 20.3 C. Initial experiments with full visible region scans showed the wavelength of maximum absorbance to be 350 nm. The buffer used throughout was 0.01 M phosphate pH 7.6. Hydrogen peroxide solutions were prepared by suitable deionized water dilutions of Merck 30% Superoxol. The actual peroxide concentration of these solutions was then determined by titration with a standardized pewnanganate solution as previously described (14). All peroxide solutions were prepared fresh daily. The dependence of and V on pH was determined using the Gilson Oxygraph and Clark oxygen e9 ectrod$aXThe 1.5 ml reaction cell was thermostated at 25 iO.2' C. The standard buffer used in the enzyme assay was 0.20 M phosphate-citrate pH 5.6. Portions of this buffer were titrated with phosphoric acid or sodium hydroxide to pH values between 5.1 and 8.3. All buffers contained 0.05% bovine serum albumin for greater enzyme stability. Ascorbic acid solutions contained 0.1% meta-phosphoric acid to ensure stability (13). A 1.5 ml volume of the appropriate buffer and 20 ul of diluted enzyme solution were placed in the oxygraph cell and equilibrated. Oxygen variation was accomplished by passing oxygen or nitrogen gas over the reaction mixture in the oxygraph cell until the desired oxygen concentration was achieved. This was followed by immediate initiation of the reaction with a fixed amount of ascorbic acid solution.

RESULTS AND DISCUSSION Addition sorption at pH 7.6. untreated and decays produced

of hydrogen

band centered

enzyme.

with

The absorption several

near

to ascorbate

hours.

stoichiometric

oxidase

produces

a AE of 900 M-lcm-l

at 350 nm with

AE was calculated

This

over

peroxide

from

a difference

band is

fully

The spectrum quantities

656

of+the

in phosphate

spectrum

formed

within

complexed

of hydrogen

a broad

abbuffer

of treated several

minus minutes

enzyme could

peroxide.

Additions

be

BIOCHEMICAL

Vol. 85, No. 2, 1978

of catalase

to the

any increase After

unchanged

system

to scavenge

rate

of decomposition

in the

complete

decay

from

its

The rate

of the initial

time

for

the complex, termine tions.

and reverse

experiment

constant

constants

equation,

[H2021

Since

for

concentrations librium

could

values.

not

sinase

(15),

Within

the

this

of enzymatic

shown

activity

could

as the

variable

seen that

the

Km for

is a random

oxygen

fluctuation

and used to de-

peroxide

concentra-

of the forward from

the

(15).

(1)

could

not be used, (1)

constants With

low peroxide

the

are

these

unknown

58.5

display

in this

equi-

constant M-is-l

parameters,

be considered used

peroxide

binding

and

the

ex-

good linearity.

to enzyme ratios

should

concentrations

initial

for the

2. The points

constant

for

tyro-

a minimum study,

value.

no inhibition

be observed.

of enzymatic

substrate.

linear

of

+ k-I

and the rate

in Figure

seen with

of peroxide

The dependence

There

are

1. For formation

enzyme tyrosinase

respectively.

equilibrium

range

the

at

of absorbance

can be obtained

approximations,

and kml,

on the deviations

which

by successive

kl

A plot

hydrogen

in equation

for

Based

was followed

were

be substituted

1.75

results

enzyme was

concentration.

to be 3.3 x104 M-l

perimental

for

to enzyme ratios

However,

s-l

complex

time

= k@-&l

was determined x10w3

complex.

by determination

process,

as demonstrated

peroxide

versus

of different

the

is the equilibrium

high

to produce

of the

shown in Figure

can be found

kobs

The term

failed

activity

concentration.

is

- At)

of kobs at a number

rate

following

peroxide

of log(Amax

The equilibrium

peroxide

of the enzyme-peroxide

of the enzyme-peroxide

a typical

values

hydrogen

the specific

spectrum,

of initial

replots

free

value.

of formation

350 nm as a function versus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity

The results is

are

insensitive of the

on pH was investigated shown

to pH over

Km values

657

in Figure

with

3. !t

using is

oxygen

easily

the region

examined.

no correlation

between

Vol. 85, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

0.03

151

0 1

O.O”o

I2345

50TIME

(minutes1 loo

150

i 1%3

2oc

A

t

:: 0.2>’

x x’

4 20

03

5

I 6

I 7

I 8

PH

The absorbance changes at 350 nm versus time showing the formation Figure 1. and decay of a hydrogen peroxide complex of ascorbate oxidase. The enzyme concentration was 29 $4 in 0.01 M phosphate (pH 7.6) at 25" C. The initial hydrogen peroxide concentration was 127 uM.

The relationship between the observed rate constant for formation Figure 2. of the hydrogen peroxide complex of ascorbate oxidase and the equilibrium peroxide concentration.

and of the Figure 3. The pH dependence of V oxidase. The line in (A) representEa$ theoretical the data. The pK for this curve is 7.8. The line method of least squares. All error bars indicate

658

for ascorbate n curve fitted to in (B) was determined by the f one standard deviation. oxygen

Vol. 85, No. 2, 1978

BIOCHEMICAL

Km and pH. On the other

hand,

the value

A titration

curve

was constructed,

fit

curve

is

of this

pK is

7.8.

must

The loss

be in its

here

In recent

between

ascorbate

and this

studies

complex

on fungal

(7,8).

acid

Gerwin,

et al.

K,,, for

ascorbate

region

crease

above

pK of about

(16)

oxidase could

pH 7.

PH. The best

an apparent

pK of

the enzyme to function.

probably

oxidase.

due to denaturation concentration

of

for

strength

have concltided

complex

similar

to those

the

of V,,,

Km for

that

effect.

the

group

site

to be constant

with

to de-

an apparent

Based on shifts

in this

pK as a

concentrations,

is

(8).

ascorbic

was found

a group

involved

that

reported

and the

V,,,

anion

with

binding

ascorbate

hand,

and polyvalent

that

intermediate.

as the peroxide

suggested this

has been demon-

a peroxide

On the other

workers

peroxide

be a catalytic

the variance

They found

pH 5 and 8.5.

ionic

and hydrogen

conceivably

have studied

8 was responsible

et al.

anionic

Gerwin,

in its

unpro-

form. Experiments

carried

out

Km for

tration

work

Since it

requires

is

the the

the effect

present is

ascorbate,

curve

ment with

involving

in the

The present

is,

at high

as a decreasing

has been implicated

pH 7 and these

of changing

the

(16!

between

result

tonated

as well

above

3A and the resulting

for

is

to have properties

Type 3 copper

in the

pH 5.5

and Rhus lactase,

enzyme has been demonstrated here

zero

in Figure

in order

below

copper,

is

rapidly

acid.

A complex strated

of its

off

in the enzyme with

form

occurs

Vm,,

line

present

protcnated

which

of the enzyme and loss ascorbic

assuming

is a group

in activity

ionized

of Vmax drops

shown as the solid

Thus there

7.8 which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also

work

with

insensitive

confirms

of pH on the the resutt

that

the decrease yields

an apparent

results

of Gerwin,

et al.

(16).

possible a proton

that

the rate

from a protein

oxidase

determining group

with

659

have been

Km for

oxygen,

between

are step

pH and a ti-

pK of 7.8,

unaffected

in good agree-

by pH but V,,,

in the catalytic

a pK of 7.8.

like

5 and 8.5.

in Vmax at alkaline

to the data

ascorbate

oxygen

the

to pH in the region

fitted

two Km's for

Km for

This

is

mechanism a rather

un-

Vol. 85, No. 2, 1978

usual

pK, being

ionize. are

BIOCHEMICAL

in a region

Based on all

an unusually

group, intriguing. it

is

placement ligands

would

ing water.

most

for

cating

type

general

sites

followed

primary

3 copper.

receive

these

that

of the monoanion Types

in oxygen Both

ascorbate

of fungal

lactase

the amount

gested

that

molecule the general applicable

oxidase

type

(21).

This

catalytic to all

It

site

on the results

at this

of hydrogen

peroxide, receive

in a similar

oxidase

mechanism "blue"

of two,

that

is

on this

present

proposed oxidases.

660

here

studies,

would

result

and the second

to the first.

approximately

twice

the size

half-molecular

two enzymes

contain

in lactase

(24,25,26).

for

and

as a catalytic

electrons

ceruloplasmin

3 copper

This

H02-,

(21), It

two "lactase-type" for

electrons

type

point.

1 and

two

pH dependence

identical,

these

point

contains

seems reasonable

are

Types

of these

at the

manner

impli-

to the following

of both

of the

transfered

can again

in such a system.

oxidase.

transfer bind

the remain-

paper

The reduction

can then

that

pK for

of a previous lead

Re-

by nitrogen

a pK of 7.8

(20),

ligands

(18,lg).

ion

of ascorbate

sites.

appears

of copper

of the

binding

is composed

some controversy ascorbate

those

very

ligands

nitrogen

in the

pK

protein

protein

proteins

shift

with

is In the

through

of copper

and ceruloplasmin

(21,22,23).

of each still

can occur

and each

subunits

is

1 and 2 copper

reduction

four

simultaneous

also

possibility

have about

to achieve

of oxygen

is

last

will

mechanism

Based

a proton

formation

intermediate.

there

A molecule

carboxyl

(17).

accepting

two electrons.

likely

weight

electron

This

an alkaline

along

catalytic

basic

of hexaaquocopper(I1)

by an essentially

to type

step

the

of this

has a pK of 6.8

possible

study,

sources

an unusually

a number

as a reductant

for

are

in the

seem quite

2 copper

2 copper

is

to cause

do not ordinarily

likely

Coordination

molecules

of this

proposal

is

often

water

would

The results

copper

molecules.

be expected

It

ion

two water

of several

the most

to copper.

to assume that

has been implicated

it

bound

RESEARCH COMMUNICATIONS

sidechains

or cysteine,

The hexaaquocopper(1:)

no more than

acid

data,

tyrosine

molecule

reasonable

with

amino

the available

acidic

or a water

where

AND BIOPHYSICAL

although

has been sug-

active as well.

ascorbate

twice

sites

per

If this

oxidase

is

may be

so,

Vol. 85, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

1. MalmstriSm,

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73, 1389-1333. R.E. and Dawssn, C.R. (1977). Biochemistry 16, 1926-1929. 20. Strothkamp, K.G. and Dawson, C.R. (1974). Biochemistry 13, 434-440. 21. Strothkamp, Freeman, S. and Daniel, E. (1973). Biochemistry 12, 4806-4810. E: McCombs, M.L. and Bowman, B.H. (1976). Biochim. Eophys. Acta 434, 452-461. 24. Deinum, J., Reinhammar, B., and Marchesini, A. (1974). FEBS Lett. g, 241-245. 25. Veldsema, A. and VanGelder, B.F. (19?3). Biochim. Biophys. Acta -293, 322-333. 26. Deinum, J. and Mnngsrd, T. (1973). Biochim. Biophys. Acta 310, 321-330.

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