Glycoenzymes: A note on the role for the carbohydrate moieties

Vol. 40, No. 1, 1970

BIOCHEMICAL

H. Pazur,

Department

Received

RESEARCH COMMUNICATIONS

A NOTE ON THE ROLE FOR THE CARBOHYDRATE MOIETIES*

GLYCOENZYMES: John

AND BIOPHYSICAL

Harvey

R. Knull

and David

of Biochemistry, The Pennsylvania University Park, Pennsylvania,

L.

Simpson'

State 16802

University

May 25, 1970

Summary: The carbohydrate moieties of the glycoenzyme, glucoamylase I from Aspergillus niger, are linked by Q-glycosidic bonds to approximately 45 serine and threonine residues, presumabIy on the surface of the enzyme molecule. The Extensive glucoamylase is remarkably stable on storage at low temperatures. oxidation of the carbohydrate residues in the enzyme by periodate markedly affects the stability of the enzyme. It is suggested that the carbohydrate moieties function as stabilizers of the tridimensional structure of the glycoenzyme, and, in turn, of the catalytic property of the molecule.

With analysis,

the many improvements

in the

it

that

has become

carbohydrate enzymes,

residues the

galactose

carbohydrate

has been

type

Most

(1).

deoxyribonuclease while

portion

suggested recent

examples

(7)

pertains

to the

molecular

level.

essential

for

An earlier the

transport

glycoenzymes (3),

An intriguing

or functions

of

proposal

(9)

of

the

enzyme

each

the

through

The term

enzyme.

bovine

of

pancreatic invertase

chloroperoxidase

(6),

aspect

the

glucose,

and yeast

carbohydrate

that

these

of enzymes

are

B (5),

linked In

mannose,

descriptive

ribonuclease (8).

of

for

and

covalently structures.

consists

alpha-galactosidase

oxidase

function

contain

characteristic

of

enzyme purification

molecular

most often

examples

include

and glucose

their

as appropriately

plant

for

many enzymes of

in arrays

(2),

earlier

amylase

as components

or glucosamine

glycoenzyme this

evident

techniques

gluco.

of glycoenzymes residues

carbohydrate cellular

(4)

at

the

residues

membranes

is

are indeed

*The work was supported in part by grants from the National Institutes of Health (AM-10822), the Corn Industries Research Foundation of the Corn Refiners Association, Washington, D. C. and the Miles Laboratories, Inc., Elkhart, Indiana. *Present Medical

address: Center,

Department of Biological Hershey, Pennsylvania. 110

Chemistry,

The Milton

S. Hershey

BIOCHEMICAL

Vol. 40, No. 1, 1970

plausible

but

difficult

carbohydrate

moieties glycoenzymes

perimental

data

role

of

tion

as

In

glycoenzyme.

the

structure

finding

of

this

of

proteolysis

(10)

communication

a

enzyme

has

made

third

a glycoenzyme but

not

from

and,

is

these the

basis

the

substantiated

proposal

of

the

that

glycoproteins

that

on

proposal

been

structure

is I,

of

specifically

suggestion

native

chain

tridimensional

glucoamylase

the

A second

polypeptide

residues, the

This

that

the

carbohydrate

stabilizers

the

experimentally.

against

(11).

the

verify

protect

presumably

the

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by advanced

new

funcmoiety

of

information

Aspergillus

periodate-oxidized

for

residues

protein

of

ex-

on

niger, enzyme

is

and

the

remarkably

stable. Aspergillus are

niger

glycoenzymes

produces

containing

form,

residues

of

glucose

and

the

present

studies.

in

subjected reaction the

glucoamylase

to

alkaline

tions lost.

of

tryptophan preparations graphy density

The

the

number

Appropriate

correction

weight

amino

factors (17)

assistance gratefully

were

purified for

amino

used

acids

(15,

acid

residues

to

on

loss

obtain

of C. Winkler acknowledged.

of the

and

111

the

A.

at

to

value

per

mole

of

be was

enzyme

carbohydrate

as

for

the

with

of

activity

was for of

exchange

found

at

condi-

hydrolyzates

this

0”

analyzed

cation

were

percentage

alkaline

then

and

Cepure

been I

enzymic

acid

values

20

has

periods

a high

and

I was

16)

data

of

by

more

mannose,

enzyme,

6-day

were

glucoamylase

based

or

and

which

glucoamylase

degraded

(14)

of

The

of of

Under

preparations

other of

4-

(13). also

(7).

mole

removal

method for

of

of

was

enzyme

centrifugation

calculating

aThe technical analyses is

native

molecular

per

highly

both

residues

galactose

protein

enzyme

analyzed

gradient

calorimetrically

the

(12)

galactose

80

a reductive

a spectrophotometric were

.a

from

and

and

reduction

effect

the

reduced by

of

Samples

reduction,

glucoamylase

approximately

2 residues

residues

the The

I with

which

of

glucose

borohydride

conditions carbohydrate

forms

mannose,

abundant

used

two

these

chromato110,000 used

from for

(Table

1).

measured

borohydride-

the

amino

acid

Vol. 40, No. 1, 1970

reduced acid

enzyme and for

hydrolysis It

serine

of

should

is

reduced

tached acid

the

values

of the

remain

1 that

gain

(18)

acids

19), are

glycine

during

threonine

is reduced

while

the

carbohydrate

to the

sugar

unchanged

in the

and a-amino

and glycosyl

reduced

relatively

balance

reduction the

(15,

a reasonable

in alanine,

borohydride

to check

stability

I were

of

for

loss

elimination

reactions,

which

(20).

the

acid

butyric

residues

alcohols

of

butyric

to o-amino

and are within

period.

water

at 4" for

found

that

changed

the

of

this

was retained. of

total

and 1 l/2

years

and 100% of

the

and the

acid are

Other

at-

amino

experimental

error

material

that

and assayed

for

activity

oxidation at the

(17)

the 6-hour

had formed less

than

or in the

enzymic

was retained

for this purpose Enzyme Institute,

112

against

distilled

procedure

this

the

sample

(21) were

a white

it

was

not

amorphous

pre-

of appropriate original

activity

in phosphate-citrate

frozen

activity,

conditions

in

of the

I was stored

of pH

and two-thirds

and assays

half

periodate

in the 6-

by a standard

sample,

on

oxidized

sample

of

residues glucoamylase

these

were

method

24-hour

glucoamylase 6 years

Under

activities

in the

purified

M sodium

residues

assays

enzyme,

showed

at 4" for

The use of periodate Kleppe (7), presently Madison, Wisconsin.

of

carbohydrate

the

in 0.02

carbohydrate

specific

denatured

of

room temperature.b

and enzyme

However

original

oxidation

dialysis

24 hours

of the

samples

by a calorimetric

When native pH 4.5

the

Following

presumably

samples

at

of

significantly.

cipitate,

periodate

as determined

influence

appropriate

or 24 hours one-third

period

a possible

enzyme,

to mild

6 hours

24-hour

for

the

subjected

approximately

b

and threonine

analysis. In order

hour

in Table

to alanine,

amino

of serine

enzyme.

to glycine

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

destruction

and the

In the

or degraded

buffer

the

and threonine

serine

5.0

the

be noted

was obtained.

the

BIOCHEMICAL

state it

for

periods

was found

in these

was first University

solutions.

that

of 94$,

l/2 98s

Quite

suggested by K. of Wisconsin,

Vol. 40, No. 1, 1970

clearly

the

native

oxidized

enzyme.

specific

role

the

It

has been

carbohydrate

residues

mately

100 carbohydrate

mately

45 points

threonine

chains

ance

of

of the

for

the

the

proposal

A glycoenzyme

the

residues.

together

with

carbohydrate

structure this

type

fungal

cell

directed

by the

appropriate

polypeptide

polypeptide

are

contains

Obviously,

do indicate

that

the

biosynthesis

carbohydrates

moieties

approxi

serine

of

the

and carbo-

calculation

to other a unique

the

chain

are

to the

this

by

approxi-

there

length

of

linked

the polypeptide

average

data

which

the

residues

the

the

conformation

carbohydrate

Nevertheless,

First,

proper

of

of

events.

into

2.2

for

linkages

with

this

is

of

the new findings

enzyme and that

l),

the

arrangement

of

the glucoamylase

(Table

for

the molecule.

of

in

second,

the

as,

in stability

of glucoamylase

of

that

chloroperoxidase

in the

residues

that

of

even when 90% of

pathways

residues

per mole

glucoamylase

intact

carbohydrases

on the basis

the known

fact

types

thesized

tors;

of

chain

conformational

the

advanced

appear

The differences

of

to a structure

would

glycoenzyme,

protein.

portion

of

enzyme

other

it

activity

and threonine

of the

the protein.

the basis

the

basis

polypeptide

for

architecture

from

residues

in the

allow

catalytic

are

of attachment

of the

23),

may be due to differences

to serine

In view

is attributable

to other of

to periodate-

tridimensional

(22,

type

The carbohydrate

bonds

in contrast

stability on the

full

conclusions

enzyme.

of

another

on the protein

(25).

e-glycosidic

acids

stability

and on the

glycoproteins

not

impart

glycoenzymes

on glucoamylase

does

literature

to retain

stable

this

in the

However,

The following

hydrate

moieties

has been removed

carbohydrate

the

carbohydrate

also

found

among various

of

is remarkably that

residues invertase.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is suggested

From reports

carbohydrate

(24)

glucoamylaae

of the

enzyme.

example,

BIOCHEMICAL

amino molecular

stability

function

data

in the

is

mainten-

of glycoenzymes. of

molecular

by the

architecture

following

chain

is assembled

messenger

RNA, transfer

chain

as determined

dissociates by the

113

is

sequence on the

probably

syn-

of molecular

ribosomes

as

RNA and associated from

bonding

the forces

ribosomes between

facand folds the

Vol. 40, No. 1, 1970

amino

acid

residues the

BIOCHEMICAL

residues

of

are attached

enzyme

molecule

via

AND BIOPHYSICAL

the

polypeptide

chain;

to the

serine

and threonine

nucleotide

diphosphate

Table Number

of amino

acid residues after reduction

and third,

.

the

residues hexose

carbohydrate on the

surface

and appropriate

glyco-

1

per mole of glucohydrolase I before in alkaline sodium borohydride BeforeC

Aspartic

RESEARCH COMMUNICATIONS

93

Afterd

and

Ratio

97

1.04

91

0.76

Threonine

ll9

Serine

132

115

0.87

Glutamic

65

66

1.02

Proline

33

32

0.97

Glycine

66

79

1.20

Alanine

90

1-15

1.28

8

4

--

55

57

1.04

Methionine

4

4

Isoleucine

32

34

1.06

Leucine

61

65

1.06

Tyrosine

34

35

1.03

Phenylalanine

32

33

1.03

Lysine

19

17

0.91

Histidine

6

6

Arginine

25

24

o.g6

Tryptophan

33

34

1.03

0

8

co

Cystine/2 Valine

(x amino CAverage dA verage

butyric of five determinations of three determinations

on four preparations on two preparations 114

of

Vol. 40, No. 1,197O

syltransferases

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(1,

maintaining the

25,

a tridimensional

structure

synthesis

glycoenzyme

even

if

such

as the mitochondria the

Also,

27).

the polypeptide cluded. the

the

Several

on the

suggestion

that

formational

to

periodate

prior

case

the

into

proposed

of the

(24)

from

molecule

residues

would

enzyme.

are

in progress the

residues

the

and determining

of carbohydrate

particles (25,

become attached course

not

the

obtaining

additional

and for

ex-

testing of

involves

by a procedure

to

manner

as well

as stabilizers

stability

valid

conformation

One experiment

the

in

glycopeptides

influence

glucoamylase

glucoamylase

equally

subcellular

is of

for

function

glycoenzyme.

from

precursor

of the

carbohydrate

is

residues

intact

for

of

in

characteristic

structure

carbohydrate

completed

carbohydrate

its

role

the glycoenzyme is

folding

of the

residues

oxidation

of

carbohydrate

folds

on the

particles

of experiments

structure

devoid

the

stability

the

carbohydrate

enzyme

chain

types

formation

or Golgi that

glycoenzyme

influencing

the

of the

possibility

In this

which

The hypothesis

26).

as

inthe con-

stripping

based

properties

the

in

of

on the

moieties.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

a. 9. 10. 11. 12. 13. 14. 15 * 16. 17.

Pazur, J. H., Simpson, D. L. and Knull, H. R., Biochem. Biophys. Co~un., J.&, 394 (1969). Catley, B. J., Moore, S. and Stein, W. H., J. m. Chem. 244 -,-

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Dey, P. M. and Pridham, J. B., Biochem. J., ly, 49 (1969). (1969). Neumann, N. P . and Lampen, J. O., Biochem., 8, 3552 (1%9). Plummer, T. H ., Jr. and Hirs, C. H. W., J. -Biol. Chem., a 2530 (1964). Morris, D. R. and Hager, L. P.. 2. Biol. Chem. 241, 1763 (1966). K. and Ball, E. M.,xh:-Biochem. Biophys., lol, Pazur, J. H., Kleppe, 515 (1963). Pazur, J. H., Kleppe, K. and Cepure, A., --Arch. Biochem. Biophys., 111. 351 (1965). a., Eylar, E. H., 2. Theoret. u 89 (1965). Gottschalk, A. and Fazekas de St. Groth, S., Biochim. Biophys. Acta @, 513 (1960). Coffey; J: W. and de Duve, C., 2. -*Biol Chem &, 3255 (1968). -*, Pazur, J. H. and Ando, T., 2. --Biol. Chem., 2& 1966 (1959). Payza, N., Rizvi, S. and Pigman, W., --Arch. Biochem. Biophys., m 68 (1969). Schram, E., Moore, S. and Bigwood, E. J., Biochem. J., a 33 (1954). Martin, R. G. and Ames, B. N., 2. a. Chem. ZI.&, 1372 (1961). Pazur, J. H., Kleppe, K. and Anderson, J?,'Biochim. Biophys. Acta & 369 (1962). Francois, C., Marshall, R. D. and Neuberger, A., Biochem. J., 8& 335 ( 1962). 115

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AND BIOPHYSICAL

J. M., Stein, E. A., Neurath, H. and Fischer, E. H., 2. m. 2j4, 556 (1959). J. B., Biochem. J., 94, 368 (1965). D. R., Carbohyd. &., L 106 (1968). J. H. and Kleppe, K., 2. Biol. w., a 1002 (1962). S., Neumann, N. P. and LGn, J. O., 2. Biol. Chem., 2&j,

(1933).

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RESEARCH COMMUNICATIONS

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Arnold, W. N., Biochim. Biophys. Acta 178, 347 (1969). Lee, T. and Hager, L. P., Federation Proceed., a 599 (1970). Schachter, H., Jabbal, I., Hudgin, R. L., Pinteric, L., McGuire, E. J. and Roseman, S., 2. m. Chem.. & 1090 (1970). -, Bosmann, H. B. and Martin, S. S., Science, l& 190 (1969). Herp, A., Liska, M., Payza? N., Pigman, W. and Vittek, J., FEBS Letters, 6, 321 (1970).

116