Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages 25-31

March 15, 1979

13C NMR STUDIES OF THE BINDINGOFSOYBEAN Michael Contribution California Received

TRYPSIN INHIBITOR

W. Hunkapiller, Michael D. Forgac, and John H. Richards

Ellen

TO TRYPSIN Ho Yu

15952, Division of Chemistry and Chemical Engineering, Institute of Technology, Pasadena, California 91125

January

17,1979

of the complex between trypsin and soybean trypsin SUMMARY : NMR studies inhibitor with l-13C-arginine and modified inhibitor with 1-13C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsinf 13C-Lys-inhibitor at pH 6.5 and 8.1 and At pH 7.1 for trypsin/13C-Argfor trypsin/13C-Arg-inhibitor at pH 5.0. inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor. INTRODUCTION Soybean hibitors

trypsin

found

lecular

of 22,000

of 10' M-l

amide

trypsin

under

support

the view

of the

simple

an analogue

the hydroxyl the complex

group exist

with

(2).

(1).

These

constant

decreasing

64 which

pH.

inhibitors

form much more stable

ST1

can be cleaved

and many other

trypsin

in-

ST1 has a mo-

an association

with

arg 63 and ile

by

observations

function

complexes

as sub-

with

the

(3).

interactions

Michaelis

tissues

rapidly

in the molecular

enzyme and the peptide

involve

complex

substrates

question

to trypsin

occurring

however,

is one of many proteinase

and plant

conditions naturally

which,

been the nature

plex

that

(STI)

Ka decreases

bond between

do normal

A central

of the

and binds

appropriate

analogues

enzyme than

of animal

at pH 8.0;

has a reactive

strate

(Kunitz)

in a variety

weight

(K,)

inhibitor

function

between

bond between binding

the residues arg

between

of the acyl-enzyme

adduct

inhibitors

has

at the catalytic

63 and ile

64:

with

carbon

in which

an ester

of arg

the hydroxyl

site

does the com-

enzyme and inhibitor

intermediate

of ser 195 and the carbonyl as a tetrahedral

of these

(4);

is

the

bond between

63 (5), group

or does of ser

T/ST1 = trypsinlsoybean trypsin inhibitor complex. STI-Arg* = ST1 with arg 63 replaced by 1-13C-Arg. STI-Lys* = ST1 with arg 63 replaced by l-13C-Lys. 0006-291X/79/050025-07$01.00/0 25

Copyright All rights

@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.

BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

195 of the enzyme has been added across the carbonyl group of the peptide bond (6,7)? In this work we have used cmr to probe this question by observing the T/ST1 complex in which arg 63 of ST1 has been replaced either with 1-13C-arg (T/STI-Arg*)

or l-l3 C-lys (T/STI-Lys*)

(8).

MATERIALSANDMETHODS Materials Carbon-13 labeled KCN (90% enriched) was purchased from Merck, Sharp and Dohme. Soybean trypsin inhibitor was obtained from Gallard-Schlesinger Chemical Corp. Lyophilized bovine trypsin (lot TRL 34B884) was purchased from Worthington Biochemical Corp. CPase B was purified from the acetone powder extract of porcine ancrease (Pancreatin Grade II and III from Sigma), as described (9). 1-l s C-Arginine (10) and l- 13C-lysine (11) were prepared by published procedures. Preparation of the Complex Between Trypsin and 1-13C-Arginine of l-13CLysine Soybean Trypsin Inhibitor. gnzymic replacement of the arg 63 residue in the reactive site of soybean trypsin inhibitor by l-13C-arginine or l-13Clysine followed the published procedure (8). Methods Carbon-13 nmr spectra A Varian XL-loo-15 nmr spectrometer operating at 25.14 M8z in the FT modewas used to record the cmr spectra. In general, spectra were recorded with the following parameters: 6000 Hz sweepwidth, 0.2 set acquisition time, and 90' pulse (at 60 usec). Proton noise decoupling was used. Digital line broadening of 6 Hz, corresponding to a sensitivity enhancement of 0.05, was applied to the FID before Fourier transformation. Temperature of the probe was 31-32OC. Difference spectra were obtained by computer subtracting a cmr spectrum of unlabeled T/STI complex taken at pH 7.2 from each cmr spectrum of the labeled complexes. (The 13C-natural abundance spectrum of the T/STI complex does not change observably in the range pH 3-8.) The difference spectra program was provided by Dr. S. H. Smallcombeof Varian Associates. Sample preparation Generally, solutions of T/ST1 complex (l-2mM) were madeup in 0.2M KC1 and transferred to a 12 mmnmr tube fitted with vortex plugs and a D20 locking capillary. The pH's of the solutions were measuredwith a Radiometer Model PM 26 pH meter. Adjustment of pH was done with either lN HCl or 1N NaOH.

RESULTSANDDISCUSSION Figure 1 shows 13C difference 6.5 and 3.0.

spectra for T/STI-Lys*

Figure 2 showsdifference

A formal difference

obtained at pH 8.1,

spectra for T/STI-Arg*

spectrum of T/STI-Arg*

26

at 7.1 and 5.0.

at pH 3.0 was not obtained, but the

BIOCHEMICAL

Vol. 87, No. 1, 1979

I

I 190

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

I

I 150

I

FROM

TMS

170 PPM

I

I 130

I

L

110

spectra of trypsin/soybean trypsin inhibitor Figure 1. The cmr difference complex (carbon-13 enriched at C-l carbon of lys-64 of STI): spectrum taken at pH (A) 8.1; (b) 6.5; and (c) 3.0. Sweep width is 2500 Hz.

13C spectrum natural

abundance

shifts C-l

of the

of the

resonances

of arginine

near

trast Lys*

should

pH where

and T/STI-Arg*

have

observed near

difference

to the single

of significant

of unenriched

and lysine

neutral

The positions

shows a peak at 177.5

spectrum

A significant ent

complex

T/STI.

for

T/STI-Lys*.

show only

a single

resonance..

l3 C resonances

of tetrahedral

a 13C chemical

shift

I lists

near

in the

the chemical as well

and T/STI-Arg*

shows two resolved

seen for

amounts

absent

and TISTI-Arg*

the T/STI-Lys*

resonance

of the

is

as for

pH.

between T/STI-Arg*

Table

T/STI-Lys*

neutral

ppm which

for adduct. that

27

for

13C resonances

At acidic

the complex

is appar-

pH both

eliminate

in conT/STI-

the presence

Such a tetrahedral

species

C-l

such as

of a substance

BIOCHEMICAL

Vol. 87, No. 1, 1979

I

I

I

I

190

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

170

PPM

Figure 2. The cmr difference complex (carbon-13 enriched at pH (A) 7.1 and (B) 5.0.

Table I: Arginine

I

FROM

Carbon-13 Chemical and Lysine, Free

for Active

the C-l Carbon Site of STI.

I!!

175.7

T/STI-Lys*

T/STI-Arg* P.P!!! 173.3,

174.9.t

I?!!

PE!!

8.1

175.2

5.0

174.8

6.5

175.2

2.9

177.5

3.0

177.6

A single resonance for T/STI-Arg* independently (D. E. Neves, J. L. Laskowski, Abstract submitted for Markley for informing us of these tion.

methane

at 94.2

170-180

ppm which

amide,

or carboxyl

which ppm (12). is

of

Is!!!!

6.8

175.5

I!!!

orthoformate

L

TMs

Shifts and in

PLP!!

6.0

ethyl

I 110

Lysine

-PB

t

I

spectra of trypsin/soybean trypsin inhibitor at C-l carbon of arg-64 of STI): spectrum taken Spectrum width is 2500 Hz,

Arginine

7.1

I 130

150

falls These

characteristic

groups

at 127.2 positions for

(13).

28

at 174.7 ppm has been observed Markley, M. E. Welch and M. 1979 FASEB). We thank Professor results prior to their publica-

ppm (12) are trigonal

or even, far

removed carbonyl

possibly, from carbons

dimethoxythe region in ester,

of

BIOCHEMICAL

Vol. 87, No. 1, 1979

Three (i)

possibilities

intact

amide

non-covalent

remain

bond

for

in a native

Michaelis

complex; complex

Michaelis

the 64-65

bond has been hydrolyzed;

as a carboxylic We identify

2.9 and at 177.6 acids

probably

acid

the bonding inhibitor

(ii)

covalent,

exist

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which

an acyl

between

trypsin C-l

present

of arginine

for

at pH 3 as arising

changes

pH 3 (a downfield

shift

of 2.0 ppm for

T/STI-Arg*

cmr studies

of many peptides

(14,lS)

that in

with the

complex

a modified

dissociates

ppm, which

suggesting covalent tween

arg

between slow

this

enzyme and inhibitor

basis,

acyl

and amides lower

field

we tentatively native

in which with

of these

T/STI-Arg*

between

pH the

for

inhibitor

with

the observed a tetrahedral

(i)

a non-

amide

bond be-

an ester

two forms

shifts

at 174.9

for

(7b)

are

in

generally On

ppm to the non-covalent signal

at 173.3

non-covalent the X-ray

might

exists

of carbonyl

esters.

Possible

29

group

of the corresponding

density

maps have been recognized

and ester

of amides

of both

adduct.

ppm and

those

account

electron

largely

at 173.3

in which These

and enzyme and the

may also

evidence

exists

of

an intact

chemical

the signal

The presence

T/STI-Lys*)

inhibitor

an amide

enzyme form

those

pH and

previous

as a mixture

Although

than

anions.

neutral

and of 1.9 for

in the same range,

assign

enzyme complex. for

fall

at pH

of carboxylic

and confirm

exists

an acyl

scale.

then

of the corresponding

and arg 63 of inhibitor.

on the nmr time

between

consistent tion

between

at somewhat

complexes (6,7)

complex

64 and (ii)

from C-l

complexes

appropriate

pH, T/STI-Arg*

63 and ile

would

T/STI-Arg*

shows two resonances

at this

of esters

complex the

in the range

ser 195 of trypsin

carbons

complex

in which

64 bond has been hydrolyzed.

that,

exchange

occur

fall

the

the arg 63-ile

Near pH 7, the T/STI-Arg* 174.9

for

at low pH at which

form in which

lysine)

ppm for

amounts

The pH dependent

accord

a non-

inhibitor

(or

at 177.5

small

we observe

and (iii)

in a

anion.

observed

with

complexes:

to the trypsin

enzyme species

the resonances

in equilibrium

is held

and a modified

or carboxylate

T/STI-Lys*

in the T/ST1

and acyl diffraction

be interpreted

ambiguities and X-ray

ppm to

studies

in

enzyme results

as being interpretaof the

complex

Vol. 87, No. 1, 1979

BIOCHEMICAL

between anhydrotrypsin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and bovine trypsin

carbonyl carbon of Lys 15 is distorted

inhibitor

pyramidally

it and ser 195 OY is too great for significant

(6b) suggest that the butthatthe

covalent bond formation.

(Another formal possibility

is that the two signals reflect

conformations of T/STI-Arg*

which interconvert

Comparison between C-l of free arginine intact

inhibitor

(174.9) shows an upfield

of free arginine as an internal lysine into the T/STI-Lys*

shifts

two distinct

slowly on the nmr time scale.) (175.5 ppm) and T/STI-Arg*

shift

with

of 0.6 ppmon incorporation

residue in the inhibitor.

complex shows an upfield

175.7 ppm in free lysine to 175.2 in the complex). upfield

distance between

shift

Incorporation

of

of 0.5 ppm (from

These values agree with

of 1.5kl.O ppm (14) and 0.8 to 1.9 ppm (15) observed for C-l

of amino acids upon incorporation At pH 5 in both TISTI-Lys*

into internal and T/STI-Arg*

positions in peptides. we observed only a single

resonance for the complex at a position assigned to the intact a non-covalent Michaelis complex with trypsin.

inhibitor

in

A recent study found s 10%

of the acyl enzyme form present in T/ST1 at pH 5.2 (16).

This is consistent

with our observation of the acyl enzyme form at neutral pH and its apparent absence in the cmr spectrum at pH 5.0 as 10% acyl enzyme is just below our observational

limits.

We observe only one resonance, characteristic T/STI-Lys*

indicating

covalently

bound amide over the pH range 5-8.

that this complex exists almost exclusively

for arginine modifies the molecular details hibitor

of an amide carbonyl,

and enzyme even though this substitution

as non-

Thus, the substitution

of the interaction causes little

for

of lysine

between inperturbation

in the thermodynamics of binding (8).

Acknowledgement: This work was supported by NIH grants GM10218 and GM16424.

30

Vol. 87, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Laskowski, Jr., M., and Sealock, R. (1971) The Enzymes, Vol. III, pp. 375-473, Boyer, P. B., Ed., Academic Press, New York. Niekamp, C.-W., Hixon, Jr., H. F., and Laskowski, Jr., M. (1969) Biochemistry, 8, 16-22. Finkenstadt, W. R., Hamid, M. A., Mattis, J. A., Schrode, J., Sealock, R. W., and Wang, D. (1974) Proteinase Inhibitors, Bayer Symposium V, pp. 389-411, Fritz, H., Tschesche, Il., Green, L. J., and Trsucheit, E., Eds., Springer-Verlag, Berlin. Means, G. E., Ryan, D. S., Feeney, R. E. (1974) Acct. Chem. Res., 7, 315-320. Hixon, Jr., H. F., and Laskowski, Jr., M. (1970) J. Biol. Chem., 3, 2027-2035. a) Ruhlmann, D., Kukla, D., Schwager, P., Bartels, K., and Huber, R. (1973) J. Biol. Biol., 77, 417-436. b) Huber, R., Bode, W., Kukla, D ., Kohl, U., and Ryan, C. A. (1975) Biophys. Struct. Mech. 1, 189-201. a) Blow, D. M., Janin, J., and Sweet, R. M. (1974) Nature 249, 54-57. b) Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H., and Blow, D. M. (1974) Biochemistry, 13, 4212-4228. Sealock, R. W., and Laskowski, Jr., M. (1969) Biochemistry 8, 3703-3710. Folk, J. E. (1970) Methods in Enzymology Vol. XIX, pp. 504-508, Perlmann, G. E., and Lorand, L., Eds., ALademic Press, New York. Pichat, L., Guermont, J. P., and Liem, P. N. (1968) J. Labelled Compounds, Vol. IV, pp. 251-255. Rogers, A. O., Emmick, R. D., Tyran, L. W., Phillips, L. B., Levine, A. A ., and Scott, N. D. (1949) J. Amer. Chem. Sot., 11, 1837-1839. Stothers, J. B. (1972) Carbon-13 NMR Spectroscopy, pp. 138-144, Academic Press, New York. G. L. (1972) Carbon-13 Nuclear Magnetic ResoLevy, G. C., and Nelson, nance for Organic Chemists, pp. 110-123, Wiley-Interscience, New York. Christl, M., and Roberts, J. D. (1972) J. Amer. Chem. Sot., 94, 45644573. Gurd, F. R. N., Lawson, P. J., Cochran, D. W., and Wenkert, E. (1971) J. Biol. Chem. 246, 3725-3730. I. E. (1977) Biochemistry I&, 2474-2478. Huang, J. S., and Liener,

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