Complexes of pyridoxal phosphate with amino acids, peptides, polylysine, and apotransaminase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 5, 1967

COMPLEXES

OF PYRIDOXAL

PEPTIDES,

PCLYLYSINE,

Frederick

Finseth

chusetts

Received

and Irwin

Institute

February

AND

pyridoxal

while

yields

a peptide

phosphate

partial

base

located

in the active

might

react polymer

site

amino

with

reacts

(7) to produce

Massa-

to form

of lysine

large

amino

in the random a Schiff

base

but does

not combine

borohydride

Since

complexes

attached

pyridoxal-P

involving

peptides

(1,4,5)

found

of polylysine.

that not

This

configuration

all the g-amino

transaminase

which

(6) but does

but not in the helical

essentially

as

is

reaction

We have

the exception

form

reduction

of pyridoxal-P

system. and small

with coil

and pyridoxal-

pyridoxal-P

(3).

resembles

with

apoenzyme

to con-

certain

in the transamination

acids

peptides

shown

Under

after

and polypeptide

with

(1).

contained

and functions

L-aspartate-

has been

molecule

of the transaminase

Polylysine-pyridoxal-P

groups

of

in several

respects

Research

were

substrates.

and Methods Chemicals

from

California

used

in all experiments

with

plied

by Mann

Research

Labs.

doxal

solutions

were

tration

per

group

acid

lysine.

Materials

of pig heart

to reduction

c-amino

combines

readily

of Biology,

2-6.1.1,

of transaminase

prior

as models

pyridoxal-P

ACIDS,

Mass.

(E. C.

groups

hydrolysis

to the

studied

serve

Department

Cambridge,

can be resolved

which

a Schiff

we have

Sizer,

transferase)

the enzyme

P (2),

W.

transaminase

amino

conditions

AMINO

7, 1967

2-oxo-glutarate two

WITH

APOTRANSAMINASE

of Technology,

Glutamic-aspartic

tain

PHOSPHATE

of 2 x 10V4 M.

kept The

Corp.

for

the exception Py ri d oxal-P,

in the dark various

Biochemical

of the polymers pyridoxamine-P,

at 4” and were amino

625

acids,

used

peptides

which

were

sup-

and pyriat a final

concen-

and polymers

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 5, 1967

were used in a wide variety affinity

of concentrations

constants (K, the reciprocal

pyridoxal-P.

in order to measure their

of the dissociation

constant) with

The solutions were buffered at pH 8 with 0. 125 M car-

bonate buffer except in those experiments from 8. 8 to 11.4 with this same buffer. at room temperature a Gary recording

in which the pH was varied Experiments

were equilibrated

for 6 minutes before spectra were determined with

spectrophotometer.

Since an isosbestic point was ob-

tained in all cases it was possible to measure K from the linear plot of the reciprocal

of the spectral change at an absorption maximum against

the reciprocal

of the amino acid, peptide,

or polymer

concentration.

Results The absorption spectrum of pyridoxal-P with L-lysine

and L-polylysine

are characterized

and of its complexes

are shown in Figure

1 Both complexes

by a maximum at about 410 rnp which is characteristic

of a Schiff base (6,8,9).

While the polylysine-pyridoxal-P

sorbs below 350 rnp in a non-specific

complex ab-

(diffuse) manner the lysine-pyridoxal-

P compound shows a sharp absorption maximum at 273 rnyJ.as well as at 410 rnp.

The polylysine-pyridoxal-P

a yellow-green

fluorescence

complex is unique, since it displays

and precipitates

from solution in the form

of minute granules. A summary of the spectra and affinity complexes with a variety in Table I.

constants (K) of pyridoxal-P

of ammo acids, peptides and polymers

is shown

It appears that all the spectra of amino acids-pyridoxal-P

resemble closely that of lysine-pyridoxal-P

(Figure

l), although the lysine

complex is a much more stable one because of the presence of the E-amino group.

The peptides (except for glycyl-lysine)

with pyridoxal-P band.

do not form Schiff bases

under these conditions and hence do not modify its 388 rnp

In some cases (tri-

and tetraglycine),

626

however,

the 310 my. band is

Vol. 26, No. 5, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PLP + Poiylysine

Wavelength

Figure

1:

Absorption alone

in rnp

spectrum

of 2 x low4

and in the presence -7 x 10 M poly-L-lysine

7.57

M pyridoxal-P

(PLP)

of 1.4 x 10m3 M L-lysine in 0. 1 M carbonate

or buffer,

pH 8.85.

replaced

by one at 322 rntq

specific

(diffuse)

of pyridoxal-P mate All they

for

there amino form

increase

and profoundly Special

Schiff

Like

but dissociates combines pH 9.6

rapidly but above

the pH titration

with

(reaction pH 10.1 curve

it,

which this

to release

is 9. 8.

conditions.

whether

its

or not

absorption

at 386 rnp

to the characterization in some

is no interaction: is exactly

respects

is stable

with

of the

resembles

to dialysis

at pH 2.2.

in 5 minutes)

627

these

polygluta-

spectrum.

pyridoxal-P

This

affinity

and for

under

decrease

complex

complete there

size

is a non-

The

pyridoxal-P,

devoted

complex,

slowly

their

and ultraviolet

has been

transaminase.

with

there

300 rnp.

formation

combine base

attention

with

complex

its visible

polylysine-pyridoxal-P aminase.

for

which

modify

complexes below

decreases

is no evidence

a typical

all peptide

in absorption

peptides

compounds

for

transat pH 5. 0

Pyridoxal-P polylysine

below

the pK calculated the pK reported

from

by Apple-

Vol. 26, No. 5, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

Addition to Pyridoxal- P

Major Band

Minor Band

‘h-w)

hw)

I

U1trBz$let

Affinity

Constant

b-w)

312 310 310 310

diffuse

Polylysine Lysine Glycyl-lysine

386 410 410 410

diffuse 273 273

- I- - - 4.63 x If7 M::: 8.62 x 1O-4 M 4.24 x 1O-3 M

Glycine+ Aspartate Diglyc ine Triglycine

410 410 388 388

310 310 310 323

273 273 diffuse diffuse

9.44 x 1.22 x - - - - -

Tetraglycine Trileucine Polyglutamate

388 388 NO

Apotrans minase (PH 5.5) %

430

None

M

- - - - -

diffuse

::: 6.66 x LO-~ M when expressed as molarity a molecular weight of 185, 000. ’

1.02 x lo-’ - - - - -

321 diffuse 310 diffuse INTERACTIaN 340

1O-3 M lo-’ M - - -

of lysyl residues assuming

With 1.0 M diglycine the 388 rn)L band changes to 410 m)*.

$ Data of Evangelopoulos

and Sizer (10).

quist and Doty (7) for the sharp transition

of polylysine

coil (below pH 9. 8) to an a helix (above pH 9. 8). pK for the interaction found to be 10.2. profile

of pyridoxal-P

The transition

extends over two pH units.

formation

from a random

For comparison the

with L-lysine

was measured and

in this case is not abrupt and the pH Hence it appears that polymer

(and not a protonated amino group) is critical,

P reacts only with the random coil form of polylysine

con-

since pyridoxalin which all the

amino groups are accessible for Schiff base formation, For purposes of comparison with pyridoxal-P the interaction

of pyridoxal

and pyridoxamine-P

628

we also studied

with polylysine,

lysine,

Vol. 26, No. 5, 1967

and certain changes

BIOCHEMICAL

other

compounds

(if any)

by dialysis. of pyridoxal-P

are

The typical glutarate,

fail

listed

in spectrum

It thus

AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

in Table

were

appears

that

important

for

observed,

or helix

form)

Hence,

although

the polylysine-pyridoxal-P

in many

and catalyze

were

when

groups

in the table. and a-keto-

added

to polylysine

or pyridoxamine-P.

complex fails

reversed

and aldehyde

pyridoxal

the complex

and non-specific readily

L-aspartate

change

pyridoxal-P,

respects,

small

listed

substrates,

(in coil

aminase

which

the reactions

any spectral plus

Only

both the phosphate

transaminase

to produce

I.

resembles

to combine

with

transsubstrates

transamination.

ACKNOWLEDGEMENT We are the National

pleased

Institutes

to report

that

this

of Health

grant

research

was

supported

by

#AM-01680.

REFERENCES I.

Jenkins, W. T., 234: 51 (1959).

Yphantis,

2.

Wada,

H.,

3.

Hughes, Acad.

R. C., Sciences

4.

Braunstein, A. E.,- Th; ?I?;; editors) 2nd edition, o . , New York (1960).

5.

Guinand, B. M., and Snell, E. IS., Comprehensive Biochemi Florkin and E.H. Stotz, editors) Vol. 15, p. 138, Elsevier co. , New York (1962).

and Snell,

D.A.

, and Sizer,

E. E. , J. Biol.

Jenkins, W. T., 48: 1615 (1962).

Chem.

I. W. , J. 237:

and Fischer, (P. D. Boyer, ap. 6, p. 113,

629

Biol.

Chem.

127 (1962).

E. H.,

Proc.

H. Lardy, Academic

Natl. K. Myrback. Press, Inc.,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 5, 1967

6.

Christensen,

H. N. , J. Amer.

Chem. Sot. s

99 (1958).

7. Applequist, J., and Doty, P., Polyamino Acids, Polypeptides, and Proteins (M. A. Stahmarm, editor) p. 161, Univ. of Wisconsm press, Madison, Wise. (1962). 8. Metzler, D. E., Ikawa, M., 2: 648 (1954). 9.

Dempsey, W. B., (1962).

lo.

~9~5~elopoulos, .

and Snell, E. E.,

J. Amer.

Chem. Sot.

and Christensen,

I-I. N. , J. Biol.

Chem. 237: 1113

A. E.,

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