Vol. 67, No. 4, 1975

BIOCHEMICAL

THE CATALYTIC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METAL ATOMS OF COBALT SCSSTITUTED

LIVER ALCOHOL DEHYDROGENASE Arthur

J. Sytkowski*and

Bert

L. Vallee

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, !Yass.

Received

November

I,1975

SUMMARY: The catalytic and non-catalytic Zn atom pairs of horse liver alcohol e dro enase (LADH) have been replaced sequentially either by 65Zn, Co or E-2 co. The Co derivatives exhibit characteristic spectra. When Co replaces the Zn atoms which exchange secondly, enzymatic activity is altered, and both imidazole and l,lO-phenanthroline (OP) significantly modify the spectrum of the catalytic Co atoms. Further, due to the removal of cobalt, the instantaneous and reversible OP inhibition of the native enzyme becomes t!me-dependent and irreversible. Jointly, these data identify the pair of metal atoms of LADH which exchange secondly under the present conditions as the catalytic one. The approach described provides a basis for the differentiation of catalytic and non-catalytic metal atoms of multichain metalloenzymes. INTRODUCTION:

The four

have been classified and replacement exhibit

but one pair spectrally in the

X-ray

catalytic

have

activities

in which of either

structure

analysis

(3-6)

(2).

cne or both

Attempts

can be shown to correspond

to that

pairs

(l),

which

to replace

the metal

of site-specific

pairs

(LADH)

enzyme species

We have now prepared

Zn by Co atoms.

of crystals

or structural,

to differentiate due to lack

either

dehydrogenase

respective

of each

Co in order

Co replaces pair

the

characteristic

of the

alcohol

and non-catalytic,

been inconclusive

replacement

liver

by Co or Cd yields

of Zn atoms with

derivatives location

into

of both

catalytic

Zn atoms of horse

atom pairs conditions

Co-LADH

of Zn atoms,and now known

from

the the

(7).

METHODS: LADH (Boehringer-Mannheim) containing 4 g-atoms Zn/mole enzyme was .-labeled with 65Zn by exchange experiments so that pairs of intrinsic atoms are sequentially replaced to yield [(LADH)65Zn2Zn2] or [(LADH)Zn266Zn2] (8), where the order of the atoms indicates the sequence of replacements thereby providing site-specific monitors for subsequent quantitative replacement with Enzymatic activity, metal content, and radioactivity were determined and LADH was dialyzed against CoCl2 Reagents were purified as described (8). *Fellow

of the Helen

tlay Whitney

Foundation

Vol. 67, No. 4, 1975

(Johnson-Matthey) Absorption spectra l,lO-Phenanthroline and imidazole from

BIOCHEMICAL

by a modification of the method of Drum and Vallee (2). were obtained with a Cary 14 recording spectrophotometer. (OP) was obtained from G. Frederick Smith Chem. Coro. Sigma Chem. Co.

RESULTS __----

AND DISCUSSION:

species

in

zinc

which

atoms

content are

and all

enzyme

is

and,

Co the

[(LADH)Co2

second,

of

I).

(Table

in

enzymatic

activity

that

the Both

at

655

~74~ in

(750)

analysis

for

of of

the of The

Co atoms.

geometry

reflect of

ligands

in

absorption both

the

Zn

the

enzymes upon

65Zn

of

from

the

of

[(LADtl)65Zn2Zn2]

[(LAD11)Co2Zn2] 2 g-atom

to

Upon

that

Co

of

the

the

second

Co/mole

and

I),

a bsorb

visible

1.9

Zn

enzyme

pair

LADH,

(Table

and

of

but

g-atom

Zn

atoms

its

a first

suggestion

one.

for

of two

x655

bands.

in

The

(7).

the

coordination

spectra

of

are

These

the

apparent

1489

mixed of

the

and

coordination ion

(9)

crystallographic of

chromophoric,

metal

>

their

geometry

examination

the

(1050)

a difference

maxima

X-ray

permit of

A655

tetrahedral

from

spectra

(c)

revealing

these

a distorted

resultant

ligands.

of

radiation

of

(75D),

position

sphere

properties

Co2Zn2]

h740

with

LADH

enzyme

[(LADli

(2050),

Co complexes

chromophoric

of

65Zn

in

mg-'

absorptivities

of

the

g-atom

= 9 min-1

the

Co replacement.

equal

maximally

geometries

native

340

zinc

exchange

loss

replaces

3.9

total

Importantly,

contains

Co then

the

intramolecular

resulting

is

intrinsic

and

4".

i.e.,

[(LADti)Co2C02]

these

those such

Zn atoms,

of

identical

site-specific

contains

[(LAD~!)CO~CO~] for

or

Zn2],

catalytic

molar

resemble

substitution

should

the

are

enzyme

pairs

Both

Co incorporation

o-f

I\A

second

pH 7,

inter-

against

and

and

at

activity

which

is

I)

similarly

specific

decreased,

nm with

existence

either

the

the

enzyme.

[(LADH)Co2Zn2]

its

is

or

stored

;(L,4DH)Zn,65

[(LADH)co~CO$

absorbance

intensities The

of

properties,

(Table

pair

dialysis

740

native

for

first

[(LADlH)Co2Zn2]

and

molar

and

pair

the

when

respectively.

second

from

of

Continued

resulting

first

parameter

65Zn

enzyme,

the

subsequent

the

isotopic

for

during

replaces

@jZn2],

Zn/mole

weeks

no evidence

hence,

their

activity

several

a reliable

dialysis,

but not

enzymatic

there

is

for

substitutes

specific for

65 Zn

Except

indistinguishable

stable

storage, Zn f

65Zn

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

catalytic complexes

and

the

nature

and

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I PROPERTIES OF LIVER ALCOHOL DEHYDROGENASES Me/LADH g-atom/mole ~65Zn

4

-

-

14

4

2

-

14

4

2

-

14

[(LADH)Co2Zn2]

1.9

0

2

14

[(LADH)Co,G"Zn,]

1.9

1.9

2

14

[(LADH)CO~CO~]

co.2

co.1

3.9

9

[(LADH)Zn265Zn2]

Imidazole

is

known

water

and forming

alter

the absorption

[(LADH)Co2C02]

to interact

with

an LADH.Zn.imidazole spectrum

shifting

absorptivity

its

mixed

while

generating

interacts

second

the

665 and 634-640

nm bands,

support

view,

of this The striking

the

are

absorption

L(LADH)Co2C02], Functionally, as instantaneous,

this

agent

with also

but

in effect

rendering

OP binding reversible

it

the

this

does not

data

suggest

(Fig. identical

of the

1490

by the

ones.

In

of [(LADH)Co2Zn2].

(OP),

a chelating

agent

Zn atoms of the native OP does not

to that

enzyme

1B).

imidazole

spectrally

1A) but markedly

Zn atoms

of

nm (Fig.

that

the spectrum

catalytic

that its

at 634-640

interpretation.

to the catalytic inhibition

Imidazole

1A) but perturbs

of l,lI)-ohenanthroline

of [(LADH)Co2Zn2J

by displacing

atoms as the catalytic

does not affect

with

atoms

now characterized

not to remove

in agreement

spectrum

These

these

effects

(7).

a shoulder

of Co atoms,

identifying

spectral

known to coordinate (10)

pair

(Fig.

metal

655 to 665 nm and increasing

the maximum at 740 nm.

with

complex

of [(LADH)Co2Zn2] maximum from

(~=2300)

__Co

the catalytic

It does not affect

enzyme

min -1 mg -1

Zn [(LADH)Zn2Zn2]

molar

*A340

alters

of [(LADH)Co2Zn2] of native

(lc)),

change that

of (Fig.

1C)

LADt( manifests

reflecting

the formation

BIOCHEMICAL

Vol. 67, No.4,1975

Figure 1: or presence Absorption imidazole. or presence or 1 mM DP:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Panel A: Absorption spectrum of [(LADH)Co2Zn2] in the absence (-) Panel B: of either imidazole or l,lO-phenanthroline (----). spectrum of [(LADH)CO~CO~] in absence (-) or presence (----) of Panel C: Absorption spectrum of [(LADH Co2Co2] in absence (-) 8 x lo- !! M enzyme: 50 mM imidazole (----) of l,lO-phenanthroline. 0.2 M Tris-acetate, pH 7.5, 4'.

Lti

1

/

2

4

6

MINUTES

Figure 2: The inhibition of [(LADlI)Co2Zn2] and [(LADH)Co2Co2] by l,lO-phenanthroline. 10 119 of [(LADH)Co2Zn2] is added to a cuvette containing 3 ml of 1 mM OP in the assay solution. Inhibition is observed "instantaneously" (lapsed time = 8 set) and does not change with time (----). Addition of Cu2+ or Zn2+ (3 mM final concentration) during the 120-Fold assay (left arrow) increases the activity within 10 sec. dilution of the solution containing enzyme and OP reverses the inhibition completely (right arrow). [(LADH)Co2Co2], 1.2 x 10-5 M, is incubated with 7 x 10-5 M OP, 0.2 M Tris-acetate, pH 7.5, 0" diluted 120-fold and assayed in the absence of OP ( -). Inhibition is time-dependent and not reversed either by addition of excess metal ions (solid curve, X) or further dilution (solid curve, XX). of a 1:l interacts

enzyme.OP with

time-dependent:

complex:

other

LADHsZn + OP 1 LADH.Zn.(OP)l.

metalloenzymes

to remove

E-Me + nOP z E + Me(OP),,

Zn,the

and for

1491

In contrast,

accompanying

LADH it

is

inhibition

irreversible.

when OP is Hence,

Vol.67,No.4,1975

it

would

would

be expected

dependent,

but

and analytical

exchanges

pair

from

of excess This

the second

pair

Further

proof

second

catalytic

[(LADH)Co2Co2] properties

to that

of the

Co sulfur

complexes than

conversely,

LADH, as is

lower

than

analogous

with

OP removes

Co spectrum

than

of

the thermodynamic The stabilities

of

site,

could

of the corresponding

complexes

to remove Co more readily

that

the

of [COG]

[Zn(OP)n)

pair.

at the catalytic

those

the stabilities

of

by OP to that

showing

systems.

such as exist

dilution

inhibition

converting

model

differs

Neither

catalytic

data

consistent

from

it

both sets of data suggest

from

thereby

[(LADH)Zn2Zn2

Zn

complexes (11).

Zn from

are much

On this the

basis,

active

site

of LADIi constitutes

a

of

observed.

Replacement chemical

of the

be expected

derives

conditions,

the OP

affected

enzyme to be the

as deduced complexes,

atoms

Thus,

of the

to be significantly

those

of metal

the

the

identify

native

Zn enzyme.

reverses

and of

present

both

there-

with

In contrast,

native

metals pair

the

and irreversible;

of the

the

We have,

definitively

inhibits

is time-dependent

of Co atoms

system

while,

under

of [(LADH)Co2Zn2],

and nitrogen

be expected

which,

hypothesis

pair

not.

Together

as that

Co

to time

[(LADH)co~Z~~]

they

and reversibly.

atoms

of this

would

lA-C),

exchangessecondly.

of metal

pair

l,lO-Phenanthroline

localizes

of Zn atoms with

instantaneous

Figs.

Zn or of other

of Co atoms which

LADII, from

I,

instantaneously

[(LADH)CO~CO~J.

OP would

(Table

2).

pair

the above expectation.

out

the mode of inhibition

nor addition

of Zn with

modification

the functional the

data

of [(LADH)CO~CO~]

markedly

higher

bear

(Fig.

and [(LADH)Co2Zn21

catalytic

of [(LADH)Zn2Zn2],

of [(LADH)Co2Co2]

secondly

inhibition

OP inhibits

the mode of inhibition

the catalytic

this

of the

of the non-catalytic

[ ( LADH)CO~CO~]. The data

pair

replacement

replacement

examined

spectral

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the mode by which

alter

fore,

that

BIOCHEMICAL

under

characteristics

instantaneous

inhibition

of the Co enzyme,to

our

Co at the catalytic very

mild

conditions

of the of the

knowledge,

site

resultant

Zn enzyme is

thus

far

1492

but which species. into

drastically The conversion

a time-dependent

unique

alters

in enzymes

of

inhibition having

under-

Vol. 67, No. 4, 1975

gone metal-assisted

BIOCHEMICAL

dechelation.

may prove

applicable

in other,

similar

to localize

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The combinations metal

of procedures

atoms and their

functional

here

employed

potential

systems.

Acknowledgements __This

work

was supported

by Grant-in-Aid

GM-15003.

References 1. 2. 3. 4. Z: 7. 8. 9.

10. 11.

Drum, D.E., Harrison, IV, J.H., Li, T.-K., Bethune, J.L. and Vallee, B.L. (1967), Proc. Nat. Acad. Sci. USA 5i-, 1434-1440. Drum, D..E. and Vallee, B.L. (1970), Biochem. Biophys. Res. Commun. 42, 33-39. Young, J.FI. and Wang, J.H. (1971), J. Biol. Chem. --246, 2815-2821. Takahashi, M. and Harvey, R.A. (1973), Biochemistr 12, 4743-4750. Drott, !H.R., Santiago, 0. and Shore, J.D.-bEF (1974 , Let!. 39 21-23. Sloan, D.L., Young, J.Pl. and Mildvan, A.S. (1975),-B,ochem,stG'14 .____ - --I 1998-2008. Br$nd&n, C.-I., Jornvall, Ii., Eklund, H. and Furugren, E.,The Enzymes, in press. Drum, D.E. , Li, T.-K. and Vallee, B.L. (1969), Biochemistry 8, 3792-3797. H.A.O. and i!illiams, R.J.P.-.(1970), in Chemical Foster, M.A., I-lill, Reactivity and Biological Role of Functional Groups in Enzymes, Biochemical Sot. Symp. No. 31, Smellie, R.M.S., Ed., London, Academic Press, p. 187-202. Vallee, B.L. and Hoch, F.L. (1957), J. Biol. Chem. 3, 185-195. Sill&, L.G. and Flartell, A.E. (1964-r-Stabmy-Constants of !letal -Ion Complexes, Spec. Publ. 17. --Chem. Sot. (London),

1493