Partial sequence of an active-site peptide from triose phosphate isomerase

Vol.

39, No. 3, 1970

BIOCHEMICAL

PARTIAL

SEQUENCE TRIOSE

AND

OF

C.

Division,

Oak

Oak

Ridge,

RESEARCH

ACTIVE-SITE

PHOSPHATE F.

Biology

AN

BIOPHYSICAL

PEPTIDE

COMMUNICATIONS

FROM

ISOMERASE*

Hartman Ridge

National

Tennessee

Laboratory,

37830

Received March 12, 1970 SUMMARY

The phosphate chloroacetol (Thr,

sequence

containing reacts,

peptide

phosphate

phosphates

isomerase

EC 5.3.1.1)

(TPI)

with

esterifies

acid

composition

(4).

In

this

total

react

specifically

mbbit

All

muscle

in the

activity

glutomyl

vicinity

communication,

DFP-treated

used

were

carboxypeptidases

Corp.

The

tryptic

digest

active-site

of CAP-modified

*Research the

Union

peptide

sponsored Carbide

(l-5).

residue

of the I report

triose

with

which

active

modified

the

AND

phosphate

TPI,

acid

sequence

and

has been of the

the

amino

determined

active-site

peptide.

METHODS

highest

purity

A and

B were

obtained

was

isolated

TPI

from

U.

of triose

Ketol-isomerase,

muscle

amino

partial

site

Chloroacetol

of rabbit

of the

by the

the

3-phosphate

loss of enzymic

an essential

reagents

with

(D-glyceraldehyde

MATERIALS

with

from

the essential glutamyl residue is Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Ala-Trp-

Ile)-Gly-Gly-Lys.

Haloacetol

(CAP)

of an active-site

isomerase, phosphate

commercially

as described rabbit

S. Atomic

Corporation.

384

from

available.

Worthington

previously

Biochem. (4)

from

a

muscle.

Energy

Commission

under

contract

Vol.

BIOCHEMICAL

39, No. 3, 1970

Edman described

degradations

were

by Konigsberg

tryptic

amino

digest

glutamyl Edman

AND

composition

of CAP-modified is Trp2,

degradations

by the

hydrolysis,

compositions

Lys,

and

Thr, the

containing

Glu,

Pro,

sequence

appeared and

initiol

identical.

tyrosine

released

Carboxypeptidase peptide

(Figure

equivalent), peptide

was

Gly2,

one

and

molar

during

the

from

isoleucine,

equivolent) latter

isoleucine,

threonine,

The

observation

present

consistent

with

glycine

penultimate residue

not

release position

of A and rapid

at the

digest.

rates

sequence

lysine usually

during decreases

of release

penultimate

a 6-hr

alonine

(7). 385

rate

molar since two

(each

Valine

approaching

rapidly

than

of these

amino

acids

Another

of release

glycine

(7).

is

observation

is that

carboxypeptidase

Glycine of the

detected

to release

more

period.

the

molar

was

A is known

position

digestion

the

active-site

(approaching

of -Gly-Gly-Lys.

the

alanine,

expected

rates.

acid

leucine,

was

and

(4).

by

degradation

position

much

Tyr I):

a single

(one

equal

alanine

amino

of the

Carboxypeptidase and

the

lysine

Glycine

at about

(Table

of only

tryptophan,

released

occupying

after

B) digestion

release

Leu,

is destroyed

as valine,

C-terminal

a tryptic

of similar

a C-terminal

did

aminopeptidose.

leucine

tryptophon,

with

peptide

by

of digestion.

consistent

alone

Ile,

residues

since

as well

threonine,

were stoges

the

a

essential

Val?,

nine

N-terminal

from

esterified,

Ala*, first

tryptophan

in the

presence

obtained

equivalents)

and

(mixture

1) resulted whose

the

of the

as being peptide

Also,

was

as

obtained

Tryptophan,which

implicated

of the

method

of a pentadecapeptide TPI

provide

was

subtractive

COMMUNICATIONS

DISCUSSION

Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Ala-. acid

RESEARCH

(6).

acid

residue

BIOPHYSICAL

performed

RESULTS

The

AND

in the

C-terminal

A

1.1

1

1

2

2

2

1

1

1

GIU

Pro

GIY

Ala

Val

Ile

Leu

Ty r

I.

2.1

2.2

1 .o 0.33 0.87

0.95 0.88

1.1

1.0

-1.2

2.1

1.1

1.1

2.1

1.1

0.95

3

Molar

4

0.82

0.30

1.0

1.1

1.4 -

2.1

1.1

1.1

OF

of amino Cyd2

0.97

ratios

DEGRADATION

0.99

0.98

2

EDMAN

0.42

0.31

1.0

1.2

1.4

2.1

1.0

1.0

0.95

5

acidsb

ACTIVE-SITE

0.35’

0.28

1.0

1.1

1.4

1.9

1.1

0.57

0.95

6

PEPTIDEa

0.7 -

1.1

0.31

0.28

0.30

0.26

1.0

1.4

1.4

1.0

2.2

0.65

0.56

0.95

8

2.1

0.65

0.55

1.0

7

0.28

0.27

1.0

r7

1.1

2.2

0.63

0.55

0.93

9

aThe CAP-labeled active-site peptide was treated initially with 0.01 N NaOH to convert the esterified glutomyl residue to the free acid. The resulting peptide (0.8 pmoles) was subjected to degradation. After each cycle, a sample representing about 0.02 pmoles of peptide was removed, hydrolyzed with 6 N HCI at 1 loo for 21 hr, and analyzed on a n Model 120C omino acid analyzer. Beck 6” Isoleucine was arbitrarily set at 1 .O. ‘Underlining indicates the amino acid disappearing at each cycle. dTryptophan and lysine were assayed only in the initial peptide.

0.90

0.99

1.0

2.0

2.1

2.2

1.0

1

Thr

1.0

1

1

Lysd

peptide

2

Initial

TTd

Amino Acid

TABLE

Vol.

BIOCHEMICAL

39, No. 3, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Time-course of amino acids released from the active-site peptide upon digestion with a mixture of carboxypeptidases A ond B. The peptide (0.5 pmoles) was dissolved in 2.5 ml of N-ethylmorpholine hydrochloride (0.1 M, pH 8.0) containing 0.15 mg of carboxypeptidase A and 0.02 mg of carboxypeptidase 8. Periodically, samples containing 0.05 pmoles of original peptide were withdrawn, neutralized to pH 2.0, and subjected to amino acid analysis on a Beckman Model 120 C amino ,acid analyzer.

The released

positions

in the

by carboxypeptidase

A peptide,

with

the

were

moiety

molar

amounts

chymotryptic

digest

of the

tryptophan

remain

occupies

to be established

peptide aheody

derived

of equivalent

Thus,

active-site

from

of Trp,

alanyl

established

Glu,

Pro,

peptide

position

10.

and

by the

modification

original

in the

of the

by CAP

Ala,

Val,

the fifteen active

the

residues

sites

which

TPI-catalyzed 3-phosphate of v

mox

most

active-site

are

striking

order

on pH,

Rose

(11)

of the

active-site

in detail

proton

and

from

a

AGWvV-X2.

isoleucine

tronsfer

has calculated

that

387

peptide possessed

(8-10).

of dihydroxyocetone

intramolecular

isolated

Ile)-Gly-Gly-Lys

been

interconversion

composed

peptide:

a characteristic

studied

intact,

on Aminex

of threonine

hydrophobic,

have

is by

feature

was

residues degradations.

still

Tyr

Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Ala-Trp-(Thr, Perhaps

Edman

by chromatography The

valyl

The

groups

12). with

by

many

mechanism

phosphate (11,

is that

and From

pK’s

ten

of

enzymic of the

glyceraldehyde the

of 6.5

dependence and

9.5

are

Vol.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

39, No. 3, 1970

involved

in catalysis.

photooxidation

have

active-site

residue

the

hydrophobic

one

cannot

exclude

a catalytically environment

glutamyl

concerning

Burton

and Waley

led with

pK 6.5

residues

in the

the

possibility

is thought

Conceivably,

the

between

plays

inactivation

prolyl

to suggest

is responsible of the

for

residue

residue, role

groups

Program,

histidine

is the

transfer.

In view

residue high.

For

example,

15). to the the

essential

proper

geometric

site.

J.

W.

ORNL,

Halleman for

helpful

and

J.

W.

suggestions

degradations.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15.

Hartman, F. C., Biochem. Biophys. Res. Commun. 33, 888 (1968). Hartman, F. C., Fed. Proc. 28, 858 (1969). Hartman, F. C., Fed. Proc., Ti press (1970). Hartman, F. C., J. Amer. Chem. Sot., in press (1970). Coulson, A. F. W., Knowles, J. R., and Offord, R. E. Chem. Commun. 1, 7 (1970). Konigsberg, W., Methods Enzymol. 2, 461 (1967). Ambler, R. P., Methods Enzyrnol. XI, 155, 436 (1967). Stryer, L., Ann. Rev. Biochem. z>5 (1968). Loi, C. Y., Hoffee, P., and Horecker, B. L., Arch. Biochem. Biophys. 112, 567 (1965). Dayhdff, M. O., Atlas of Protein Sequence and Structure, Vol. 4, Chapter 7, p. 47. Silver Spring, Maryland: National Biomedical Research Foundation (1969). Rose, I. A., Brookhaven Symp. Biol. 12, 293 (1962). Rose, I. A., O’Connell, E. L., and Mortlock, R. P., Biochim. Biophys. Acta 178, 376 (1969). Burton, P.M., and Waley, S. G., Biochem. J. 100, 702 (1966). Donovan, J. W., Laskowski, M., Jr., and ScheraE H. A., J. Amer. Chem. Sot. g, 2154 (1960). Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C ., and Sarma, V. R., Proc. Roy. Sot. Ser. B Biol. Sci. 167, 378 (1967). 388

and

in TPI,

is in a hydrophobic

in establishing

to Drs.

that

is adjacent

of the active

I am grateful

acid

abnormally

(14,

which

iodoacetic

glutamyl

of lysozyme

a pK of 6.3

by

proton

essential

of its pK being

an important

Anatomy

of TPI

(13)

vicinity

to have

certain

of the Molecular Edman

and

glutamyl

and

ACKNOWLEDGMENT: Eveleigh

on the

functional

residue,

orientation

Studies

of