Thermolysin: A zinc metalloenzyme

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

A ZINC METALLOENZYME

THERMOLYSIN: Samuel A. Latt*,

Barton

Holmquist-

and Bert

L. Vallee

The Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts

Received

August

19, 1969

Metal analyses and inhibitor studies have shown that thermolysin, a neutral protease from B. thermoproteolyticus, is a zinc metalloenzyme. The relevance of this fiaing to the active site characteristics of other bacterial neutral proteases and to those of alkaline proteases is considered. A catalytically

INTRODUCTION: active

sites

of a number of proteolytic

endopeptidase

specificity

chemical

(5,6,7,8)

as to its

demonstrate

metal

that

content

thermolysin

and Calbiochem

crystallized high

three

pH, followed

is a zinc

Thermolysin

or more times

before

+I-

Fellow

+Fellow

of the National

Institute

of the National

Cancer

use,

This either

at neutrality

the metal

by Grant-in-Aid of the Department

333

though

no

studies

material

was re-

by dissolving (2)

at

or by dissolving

to low ionic

content

(Osaka,

strength

and the specific

act-

GM-15003 from the National of Health, Education and Welfare.

of Arthritis Institute

(6),

from Diawa Kasei

pH and dialyzing

Both

crystallization.

as

stabilized

The present

California).

by recrystallization

of Health

as well

metalloenzyme.

was obtained

(Los Angeles,

an

recently

The enzyme is

as yet.

at the

Thermolysin,

properties'(3,4),

are on record

+ This work was supported Institutes

(1).

has been purified

and kinetic

the enzyme in 5 M NaBr at neutral to promote

enzymes

by EDTA and l,lO-phenanthroline

MATERIALS AND METHODS: Japan)

atom has been found

have been described.

by Ca 2+ (2) and inhibited data

zinc

from -B. thermoproteolyticus,

(21, and some of its its

essential

and Metabolic

Diseases

Vol. 37, No. 2, 1969

ivities

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of material Metal

obtained

analyses

(9) and by atomic the basis muffle

on the

ing

Values

samples

amide

observed

0.01

M Ca2+ , pH 7.5,

p = O.lM

tion

(10 half

in

ed as k = kobsd/[E] lcm = 17.65 OD280

for

Inhibition

in M-'min

studies

of stock

solution

of substrate

furylacryloylglycine

All several extraction.

and appropriate

and inhibitor. after

(methanol-CHC13,

Acid

1:9)

use.

cleaned precautions

were proportional

containing

The first

readings

weight

Products

by the

of absorbance of the hydrolysis, by thin

silica grade

Buffers

were rendered

metal-free

glassware

or nalgene

tubes

37,500(3).

an equilibrated

were of reagent

334

are expressbased on

were identified

metal

to comple-

were performed

on fluorescent

to prevent

lives.

and were followed

agents

Km,

in 0.05 M Tris,

cuvette

amide,

and chemicals

before

constant,

Rate constants

enzyme addition.

and leucine

inhibitors times

binding

utiliz-

California).

two half

of an enzyme of molecular metal

for

the substrate

enzyme concentration

enzyme to a reaction

one minute

chromatography

with

rate

at 25'

with

and

of furylacryloyl-

at least

experiments.

a 1% solution

addition

were taken

-1

material

communication),

below

were performed

(NaCl)

several

for

order

Reactions

to enzyme concentration.

personal

of 1 mM, well

first

were similar

Company, Los Angeles,

order

an electric

based on a procedure

at 345 mu on hydrolysis

first

on

enzyme (3).

Feder,

Chemical

in

analyses

on native

spectrophotometrically

were

lives)

of zinc

of the

(12;

spectrography

Data are reported

spectrometry

concentrations

the

emission

the same.

were ashed at 450'

The results

(Cycle

of hydrolysis

of kobsd,

which

coefficient

substrate

rates

(10,ll).

in absorbance

glycyl-L-leucine

all

spectrometry

of -B. subtilis

the decrease

are virtually

by spark

absorption

was assayed

procedure

both

analysis.

extinction

protease

At initial

for

by atomic

Activity neutral

absorption

before

when performed based

were performed

of dry weight

furnace

by either

gel

layer (Eastman).

or recrystallized by dithizone

were used throughout

contamination

were

observed(1

1).

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

RESULTS:

The metal

ed thennolysin ug/g

zinc,

other

3800 ugfg

metals

found

in solution

incubation Figure

of five

are shown in Table

to reaction

Preparations

mixtures

1 shows the effect

throline,

thioglycolic

pKI for

2,2'-bipyridine,

cyanide

were

the

about

2000

concentration

of

3.1,

these

agents

of increasing acid

gave identical

Spectrographic

The addition

Analyses

of l,lO-phenanValues'

mercaptoacetic

respectively.

pre-

results.

on enzyme activity.

mercaptoethylamine, 2.4 and 2.2,

zinc

Prolonged

concentrations

and imidazole

up to 0.08 M.

known to bind

instantaneously.

Table Emission

of recrystalliz-

contained

of agents

hydrolysis

of the enzyme with

at concentrations

I.

preparations

on the ave.rage,and

calcium

inhibited

3.4,

different

was insignificant.

The addition ions

analyses

Azide of zinc

did

ions

acid,

and

not

inhibit

to enzyme solu-

I. of Three

Times Recrysallized

Thermolysin Metal

J -Zn

Content

(ug/g)

-Ca

-Fe

&

E

*

1

Sample 1

2100 + 200

3400

*

2

2000 5 300

3900

310

50

*

3

2000 2 300

3500

350

100

20

4

1850 + 200

4000

80

25

2

5

1900 Ifi 300

4400

100

20

*

7 Includes atomic absorption ments were performed at least metals were measured at least are the averages.

measurements. All zinc measurein quadruplicate; all other in duplicate. The values given

* = Not detected; also Cd, Co, Cr, Mg, Mn, MO, Ni, Pb, Sr. Copper was not determined due to the method employed (9). 335

of

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1: Effect of Metal Binding Agents on the Activity Thermolysin. Initial substrate concentration was 1 m M in M Tris-Cl, .Ol M CaClz, and NaCl to ionic strength of 0.1 25'. Activity is expressed as 100 times the ratio of the inhibited rate constant, kI to the control kc, determined described under Materials and Methods. kc under the above conditions is 9 -+ 1 x 105M-1min"~1.

tions, in

complete

which with

fully

inhibited reactivation.

reactivated the effect Dialysis

by l,lO-phenanthroline

of excess versus

three

0.05 M HEPES, pH 7, 0.4

zinc

ions

changes

M NaCl,

as

or 2,2'-bipyridine,

Concentrations

the enzyme completely,

of .05 at

of zinc caused

on the native of 0.001

ions,

resulted

in excess

inhibition,

of those

in agreement

enzyme.

M l,lO-phenanthroline,

0.01 M CaC12 for

eight

hours

each,

0 MOLES kn /37,500

g2ENZ.

to Apothermolysin upon Figure 2: Return of Enzymatic Activity Apothermolysin, prepared as described in Readdition of Zinc. the text, contained five per cent of the initial zinc content, and possessed six per cent of the activity of the native enzyme. Activity data are expressed at the ratio of kZn, the rate observed with zinc added to the apoenzyme, to k c, the rate of the native enzyme. 336

Vol. 37, No. 2, 1969

followed each,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by dialysis reduced

of the

enzymatic

control.

ed by the

against

Moreover,

zinc

the

addition,

at least

a zinc

from

for

(9,14),

number of chelating

ions

Thus, zinc. (Figure

is

essential

agent

2).

agents,

While metal

ions

for

single

enzymes

has been reported subunit the

zinc

structure. content

the

but

it

1).

from

IIB

and was inactive. and removal

containing

is not

calcium,

of experimental

error.

in catalytic

activity.

of the number of with

molecular

known whether

at present (Table

337

it

would I)

relative

weight

or not

of zinc

ease,

metalloof 37,500

the enzyme has

seem advisable

or moles

of

activity

when i'nvest'igatingmultichain

a preliminary

zinc

of the

atom restores

the stoichiometry

ions

thereby

dialysis

interaction

directly

gram A

Further,

zinc

has been established

in vg/gm

contains

enzyme.

inhibitors,

contain

in buffers

proteins

molecule

Hence, either

not

seem involved

For thermolysin (3),

for

have

thermolysin

and group

(Figure

the limits

have been problematic

(15).

at

2000 ug per

of the

of the metal

within

chain

about

resulting

did

and assayed

do not

atoms per protein

such efforts

enzyme zinc

the reincorporation

that

transition

the enzyme through

When stored

calcium

upon

consistently

function

thermolysin

The product

inhibits

establish

averaging

known to bind

inhibit

the apoenzyme was inactive Thus,

grams

in such buffers

thermolysin

to the

l,lO-phenanthroline

Further,

37,500

of metalloenzyme

data

Recrystallized

the inhibition.

this

per

to 5%

reestablish-

of activity

the identification

compete with

enzyme against

content

was completely

on storage

and the present

effectively

effectively

reversing

hours

two weeks.

The metal

in solution,

eight

6% and zinc

by regeneration

apoenzyme was stable

metalloenzyme.

I).

than

for

1 gram atom of zinc

1700 to 2300 pg per gram of zinc,

(Table

of buffer

2, activity

as judged

The criteria

been delineated is

to less

of approximately

of apoenzyme.

DISCUSSION:

activity

changes

As shown in Figure

addition

5" C for

three

to express per

375500

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

grams of thermolysin structure

(Figure

of thermolysin

is

enzyme can be assigned in view ently

of the in

there

all

is

large

enzyme,

(15)

and,

metal

for

hence,

definition

as to the

peptidases

and their

Similarly,

the neutral

proteases.

throline,

is

inhibited

which

well

reveal

peptidases of their

residues

(21).

active

bacterial differ sites,

enzymes of mammalian characteristic themselves preservation

both

of a zinc

neutral

study

proteolytic by the carboxy-

and &

zinc

megatherium

megatherium,mega-

may also

proteases

at least

be a metallofrom bacteria

contrasting

have been found

in pH optimum

from

and l,lO-phenan-

protease

contain

(16).

of bacterial

from L

features,

with

two classes

and the detailed

seryl

chemical

whi.le

mechanism properties

33%

residue

some exopeptidases of action. at the

of

chemistries

Here,

a DFP reactive

alka-

to have DFP

of the proteolytic

endopeptidases

purposes

more precise

and that

(18)

of neutral

generally

atom in their

of distinctive

(17)

That

of

rigor-

in some ways reminiscent pancreas.

of certain

function

aminopeptidases

A. oryzae

strains

in

enzyme.

by EDTA, 2,2'-bipyridine

which

which

await

examples

such common chemical

certain

Thus,

must

enzymes as exemplified

this

Though

or both

number of mammalian

proteases.

proteases

seryl

structural,

of the

now constitute

that

2+

also

consist-

described.

of Ca

from -B. subtilis

a systematic

Thus,

bacterial

reactive

suggested

here

of calcium

S.griseus,

neutral

enzyme (20).

line

(16)

imperative

have been found

and by certain

protease

contain

theriopeptidase,

role

precursors

appear

of zinc/

have to be investigated

architecture

-B. aeruginosa, also

will

to be zinc

-B. thermoproteolyticus

which

functional,

an increasing

years,

would

participation

such possibilities

of the protein

a stoichiometry

of thermolysin

can serve

enzymes have been found

might

of calcium

of the molecular

In recent

(19,20)

This

the direct

atoms

investigation

before

definitively.

of the preparations

Conjectures

ously.

A detailed

required

amounts

no evidence

the

2).

Could active

is avail

the sites

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

two types contain

of proteolytic information

and mechanisms and tertiary e.g.

Ca

2+

structure

elucidation

It

both

could

to the evolution

of the details

seryl studies of these

of their

changes

dependent

to modulate

forms

of enzymatic

be that

those

and the active enzymological

and higher

the evolution

including

, have been employed

Comparative

relevance

concerning

of action?

atom in one class iant.

enzymes in lower

secondary

such as,

to which

such lines

classes

mechanism

in primary,

in the other

along

specificities

upon ions

functions

residue

of life

the

zinc

are invarmight

have

of enzymes and the

of action.

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

Vallee,

B. L. and Wacker, W. E. C., in "The Proteins," Vo1.5, 8. Neurath, Ed., Academic Press, New York, New York, in press. Tech. do, 346 (1962). Endo, S., J. Fermentation Chem. 241, 5919 (1966). Ohta, Y., Ogura, Y. and Wada, A., J. Biol. Chem. 242, 509 (1967). Ohta, Y., J. Biol. Matsubara, I-l., Singer, A., Sasaki, R. and Jukes, T. A., Biochem. Biophys. Res. Commun. 21, 242 (1965). Morihara, K. and Tsuzuki, II., Biochim. Biophys. Acta 118, 215 (1966). I-I. and Oka, T., Arch Biochem. Biophys. 123, Morihara, K., Tsuzuki, 572 (1968). A. and Sasaki, R. Biochem. Biophys. Res. Commun. Matsubara, II., Singer, 2, 719 (1969). Vallee, B. L., Adv. Prot. Chem. lo, 317 (1955). Fuwa, K. and Vallee, B. L., Anal. Chem. 35, 942 (1963). P., McKay, R. and Vallee, Fuwa, K., Pulido, B. L., Anal. Chem. 36, 2407 (1964). Res. Commun. 2, 326 (1968). Feder, J., Biochem. Biophys. Thiers, R. T., in Methods of Bdochemical Analysis, Vol. 5, D. Glick, Publishers, Inc., New York, New York, p. 273 Ed., Interscience (1957). Vallee, B. L., in "The Enzymes," P. D. Boyer, H. Lardy and K. Myrback, Eds., Vol. 3, Academic Press, New York, London, p. 225 (1960). Drum, D. E. and Vallee, B. L., in press. Vallee, B. L. and Latt, S. A., in Dress. McCann, J. D., Tsuru, D. and Yasunobu, K., J. Biol. Chem. 239, 3706 (1964). Morihara, K., Biochem. Biophys. Res. Commun. 26, 656 (1967). Millet, J. and Acher, R., Biochim. Biophys. Acta 151, 302 (1968). Sot. Chim. Biol. 5l, 61 (1969). Millet, J., J. Bull. Vallee, B. L. and Riordan, J. F., Ann. Rev. Biochem. 2, 733 (1969).

339