p -Hydroxybenzoate hydroxylase: Conformational changes in crystals of holoenzyme vs holoenzyme-substrate complex

BIOCHEMICAL

Vol. 34, No. 1, 1969

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E-HYDROXYBENZOATE HYDROXYLASE: CONFORMATIONAL CHANGES IN CRYSTALS OF HOLOENZYME -VS HOLOENZYME-SUBSTRATE COMPLEX Keiji Department

Received

November

Yano,

of Agricultural University

hydroxybenzoates

some detail.

and requires

shown

to form 1966).

al., putida tion

addition

complex

FAD but are

attributed

Pseudomonas

holoenzyme-substrate

complex. with

in the visible

absorption

and differences

region

suggest

in the

followed

previously

reported

Protein from

(Yario

substrate

change

in the

protein.

the holoenzyme

of 68,000.

was determined

P. desmolytica

et al.,

1966).

substrate

according

procedures

in the ultrabinding. NADPH oxidato Kalcker,

IAM 1123 cultured All

in addition.

in sedimentation

dispersion

on the

and the

The molecule

shift

rotatory

hydroxy-

on ultracentrifuga-

by the -p-hydroxybenzoate-dependent

at 340 my-

The enzyme was prepared

by the

1 mole of the substrate

change

The absorp-

region

a slight

et

Pseudomonas

1966).

are homogeneous

was

(Katagiri

of p-hydroxybenzoate

weight

optical

1965)

from

et al.,

namely

spectrum,

a conformational

The enzyme was assayed tion

a molecular

1 mole of FAD and the complex

coefficient violet

Both forms

et al.,

FAD and salicylate

forms

in

FAD as the prosthetic

hydroxylase

IAM 1123,

hydroxylases

and investigated

(Yamamoto

to a conformational two crystalline

and electrophoresis

Changes

donor

in the visible

desmolytica

two bacterial

contains

NADPH (Hosokawa

changed

lase

contains

which

p-hydroxybenzoate

slightly

we have prepared

studied,

of apoenzyme,

requires

Recently,

tion

of Agriculture Japan

to homogeneity

NADH as the electron

and were

from

purified

hydroxylase

The crystalline

spectra

Chemistry, Faculty of Tokyo, Tokyo 113,

have been

a ternary

contains

and Kei Arima

monooxygenases

Salicylate

group

Higashi

1, 1968

Among the flavoprotein for

Naoki

for

1947.

by the method the preparation

Vol. 34, No. 1, 1969

were

BIOCHEMICAL AND BIOPHYSKAL

done at 0 - 5*C.

M succinate

buffer,

were

pH 6.0,

mercaptoethanol. stated.

Cells

containing

The same buffer

A clear

supernatant

at 100,000

sulfate

and centrifuged

to remove

through

a DEAE-Sephadex

A-25

(0.35

volume

x g for

- 0.60

of the buffer,

fractions

were

enzyme was eluted eluate

with

about

excess Fine

yellow

tallization

was done

recognized

G-25

column

sulfate

to remove

a slightly

holoenzyme

reduced

which

concentrations

were

enzyme forms

phoresis.

A typical

molecular (Yphantis, activity

weight

were

that

the

was calculated

Since

and the

substrate. Recryshere

were

in Fig.

through

addition

a Sephadex

of ammonium

was kept

in a desiccator

more than

3 days.

The crystals

the

increase

and the protein

partial

to be 68,000

is summarized volume

by the short

specific The number

2

(Fig.

activity

of

of the 2).

on ultracentrifugation

specific

1.

in a minimum

solution

with

the

The enzyme

obtained

and passed

procedure

to be 2700.

washing,

chloride,

of the

was dissolved

appeared

the maximal

Active

and are shown

After

homogeneous

purification

was estimated

1960).

rods

in a minimum

a few minutes.

complex

ammonium sulfate

crystalline

On the assumption

for

frac-

to be a slightly

The crystals

substrate.

yellowish

of both

within

complex

pressure

was passed

After

M sodium

in the presence

the

it

of protamine

filtration.

was brought

appeared

saturation,

of the

ammonium sulfate.

mercaptoethanol

the bound

to make a 0.55

under

0.15

same manner.

containing

gel

column.

with

holoenzyme-substrate

of the buffer

Then

A-50

to be the holoenzyme-substrate

The crystalline volume

in the

otherwise

0.1 volume

G-100

concentration,

crystals

unless

was dissolved

containing

of ammonium sulfate

and 1 mM

by an ammonium sulfate

by a Sephadex

by precipitation

needle-shaped

with

followed

in 0.017

centrifugation

the precipitate.

the buffer

oscillator

steps

by the

The precipitate

a 3 W protein

saturation

all

was treated

on a DEAE-Sephadex

was concentrated

solution,

obtained

column,

followed

applied

a sonic

was used for

30 min.

satn.).

with

0.1 mM p-hydroxybenzoate

fluid

sonicate

tionation

disrupted

RESEARCH COMMUNICATIONS

Both and electro-

in Table

I.

was 0.75

ml/g,

column

method

was 40, the molecular

of FAD molecule

was computed

BIOCHEMICAL

Vol. 34, No. 1, 1969

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig.

Fig.

to be 0.9 by assuming that for

of free NADPH.

FAD. Optimun

that

the molar

The -Km values pH was 8.2

extinction

were to 8.6.

3

25 PM for

1. Crystalline substrate

holoenzymecomplex.

2. Crystalline

of bound

holoenzyme.

FAD is

p-hydroxybenzoate

The enzyme reacted

the same as and 40pM

specifically

Vol. 34, No. 1, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

I.

Purification

of p-hydroxybenzoate

Treatment Crude extract Protamine sulfate treatment Ammonium sulfate frac. Sephadex G-100 gel filt. DBAE-Sephadex A-50 column Holoenzyme-substrate complex First crystallization Second crystallization Holoenzyme Second crystallization

with

NADPH and not with

benzoate, tion.

however, Very

observed

with

Total protein

Total activity

Specific activity

yield

ml 1,500 1,650 50 130 100

w 40,000 29,900 4,210 895 160

units 12,000 11,850 9,700 7,650 6,000

units/ml 0.3 0.4 2.3 8.5 37.5

% 100 98.5 80.5 63.2 50.1

3 3

123 97

4,900 3,900

39.8 40.2

40.7 32.3

2

76

3,060

40.0

25.4

highly

specific

NADH, and was also

activities

spectra the

of which

were found

and 2,4-dihydroxybenzoate Absorption

Volume

the concentration

low

(0.9 of both

spectrum

hydroxylase

with

above

to p-hydroxy-

1 mM showed

3,4-dihydroxybenzoate

an inhibi(1.4

%)

%). forms

of the

are

complex

shown in Pig. at 365,

380,

3.

Shoulders

were

430 and 475 rnp, while

Visible absorption spectra of both forms. Concentrations of each enzyme solutions were 260 m)rmoles (19 mg) in 3 ml, at pH 6.0. -; the holoenzyme, -----; the holoenzymesubstrate complex. Hitachi 124 double beam spectrophotometer.

the spectral titration ratio

curve

of the holoenzyme

of the holoenzyme of the holoenzyme

was rather

by E-hydroxybenzoate

: the

substrate

smooth,

Spectrophotometric

at 385 rnr showed

was 1 : 1.1 at pH 6.0.

4

that

the

When the

BIOCHEMICAL

Vol. 34, No. 1, 1969

holoenzyme

was excited

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at 375 my, a sharp

having

a maximum at 520 rnp was observed.

erable

quenching

of the fluorescence at this

showed

that

above mentioned

strate

complex

ratio

while

was 1 : 0.9.

formed

spectrum

binding,

a consid-

the substrate

Fluorometric

wave length.

was specifically

emmision

On the substrate

occured,

not show any emmision the

fluorescence

titration

Thus

by an equimolar

itself

did

at 520 rn+

the holoenzyme-sub-

holoenzyme

and sub-

strate. Sedimentation of the complex

coefficient, 5.0 S.

performed

in the

two cells

containing

volume

a single

cient

same run

appeared peak.

time really

using

in Schlieren

exists

This

indicates

between

the

larger

pattern case,

was 5.1

than

that

that

and also

centrifuge

with When the

of the complex, and finally

in sedimentation demonstrates

two

fused

of two boundaries

a difference

two forms

were

respectively.

became closer the distance

S and that

experiments

UCA-1A analytical

and the complex,

was slightly

In the reverse

elapsed,

a Hitachi

the holoenzyme

of the holoenzyme

boundaries

with

S20 w, of the holoenzyme , To confirm this small difference,

that

into

increased coeffia small

3a

Fig. 2a ‘b

4. Ultraviolet dispersion hydroxylase.

optical rotatory of p-hydroxybenzoate

Cell chamber was kept an anaerobic condition with nitrogen gas at a slightly positive pressure. The concentration of the enzyme was 0.12 to 0.0022 % at pH 6.0, 22-25'C. The mean residue molecular weight of 115 was employed.

2 IO

-; ------;

the holoenzyme, the holoenzymecomplex.

Japan Spectroscopic ORD/W-5 recording

5

substrate

model polarimeter.

Vol. 34, No. 1, 1969

but

distinct

conformational

An attempt aqueous

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the optical

The curve

effect

since

specific

holoenzyme

the

(Cassim

et al.,

1967).

the case of the

complex

from

ed unchanged.

The results

coil

difference

with

that

the

wave length

hindered

shift

% helix

suggest

that

binding

in the p-structure

Crystallization

region

content

could

a-Helix

content

of the complex

in

remain-

change

and/or

might

in a

was observed

conformational

This

of Cotton

to be -8190

of the curve

a small

in

the measurement.

was calculated

to a 47.6

A slight

forms

might

in the random was rather

easy

also

be due to the

was first

reported

in the conformations.

acid

of an enzyme-substrate

oxidase

(Yagi

(molecular

since

it

et al.,

oxygen).

enzyme-substrate

oxygen,

4 has a trough

shorter

(m')232

of the holoenzyme.

Crystallization

acceptor

in the

of both

in the ultraviolet

208 to 225 rnp , however,

of the protein.

as compared

structures

dispersion

corresponds

on the substrate

region

D-amino

value

internal

absorption

value

This

be induced

peak

rotation

was caused.

shown in Fig.

strong

1963).

protein

the rotatory

The positive

not be determined,

(Fasman,

the

of

at 232 mp.

The corrected

of the protein

was made to clarify

solutions

region.

change

1964)

complex

in the absence

of the

electron

In the case of p-hydroxybenzoate

complex

was easily

can not react

with

crystallized

the substrate

even without

with

hydroxylase,

in the

presence

electron

donor,

of

NADPH. A rather D-amino

acid

et al.,

1967).

large

reduction

oxidase

It

Very

small

(Yankeelov

a complex

benzoate,

shift

was found

that

enzyme which

protein

moiety.

The details

The authors wish Paul's University,

1965),

had not drawn

p-hydroxybenzoate might

with

was induced

et al.,

of ultracentrifugation

present

St.

coefficient

on forming

phosphoglucomutase two runs

in sedimentation

be a result will

plays

was reported an inhibitor

by the substrate however,

in this

a role

in the case of case,

separate

as an activator

in a subsequent

to express their sincere thanks to Prof. discussion. Tokyo , Japan for his useful

6

(Yagi

a conclusion.

of the conformational

be described

with

change

of the of the

paper. T. Kimura, The

Vol. 34, No. 1, 1969

BIOCHEMCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

authors also indebted to Dr. E. Ichijima and Mr. K. Hayashi, Central Research Institute, Kikkoman Shoyu Co., Noda, Japan, for their kind help in ultraviolet optical rotatory dispersion measurements. References Cassim, Fasman,

J.Y. and Yang, J-T., Biochem. Biophys. Res. Comm., 26, 58 (1967). G.D. in "Methods in Enzymology" Vol. 6, Pd. by Colozck, S.P. and Kaplan, N.O., Academic Press, New York (1963), p. 928. Hosokawa, K. and Stanier, R.Y., J. Biol. Chem., 241, 2453 (1966). Kalcker, H.M., J. Biol. Chem., 167, 461 (1947). Katagiri, M., Takemori, S., Suzm, K. and Yasuda, H., J. Biol. Chem., 241, 5675 (1966). Yagi, K. and Ozawa, T., Biochim. Biophys. Acta, 81, 29 (1964). Yagi, K., Naoi, M., Harada, M., Okamura, K., Hidaka, H., Ozawa, T. and Kotaki, A., J. Biochem. (Tokyo), 61, 580 (1967). Yamamoto, S., Katagiri, M., Maeno, H. and Hayaishi, O., J. Biol. Chem., 240, 3408 (1965). Yano, K., Morimoto, M. and Arima, K., Agr. Biol. Chem (Tokyo), 30, 91 (1966). Yphantis, D.A., Ann. New York Acad. Sci., E, 586 (1960).