Phosphorylation of alkaline phosphatase (E. coli) with o- and p-nitrophenyl phosphate at ph <6

Vol. 28, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHOSPHORYLATION OF ALKALINE PHOSPHATASE (E. coli) WITH o- AND p-NITROPHENYLPHOSPHATE AT pH ~6~ Wilmer K. Fife2 James Bryant Conant Laboratory Harvard University Cambridge, Massachusetts Received May 2, 1967 The phosphorylation

of alkaline

phate and glucose-6-phosphate

phosphatase

(E. coli)

has been studied

by several

1961, 1962; Schwartz and Lipmann, 1961; Levinthal Aldridge ever,

et al.,

1964; Pigretti

did not permit

phosphorylating number of active behavior

direct

sites

workers

These experiments,

of the reaction an accurate

on the enzyme nor a clear

(Engstrom,

1962; Schwartz,

et al.,

1965).

phos-

196 3; how-

between enzyme and determination

understanding

of the

of enzyme

in acid media.

phosylation

the preliminary

of alkaline

of lo+

determining

process.

in which phosphosphate3 at pH

methods.

This technique sites

At enzyme con-

amounts of nitrophenol

enzyme during a very rapid reaction

the number of active the interaction

of experiments

by spectrophotometric

to 10m5 g detectable

along with phosphorylated the steady-state

results

phosphatase by o- and p-nitrophenyl

was observed directly

centrations

tatively

observation

agent nor did they provide

This paper reports

3.9-5.7

and Milstein,

by inorganic

provides

are produced which precedes

an excellent

means of

on the enzyme and of studying

between inorganic

quanti-

phosphate and enzyme.

lThis work was supported by a research grant to Prof. the U. S. Public Health Service (GM04712).

F. H. Westheimer from

*National Institutes of Health Special Post-Doctoral Fellow (l-F3-GM-23,392-01) on leave from Muskingum College, New Concord, Ohio, 1965-66. 3Abbreviations:

o-NPP - Disodium o-nitrophenyl p-NPP - Disodium p-nitrophenyl

309

phosphate, phosphate.

Vol. 28, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL

The data suggest but do not establish (1)

Alkaline

phosphatase

per dimeric (2)

unit

the following

(E. coli)

contains

conclusions:

two principal

active

sites

(M.W. 86,000).

The number of active

sites

dent of pH (3.9-5.7),

determined

but it

(o-NPP or p-NPP) and/or sites

RESEARCH COMMUNICATIONS

experimentally

is indepen-

is somewhat dependent on substrate

substrate

concentration.

(The number of

found was 1.7 * 0.1 at [o-NPP] = 5 X 10D5- 2 X 10q4 Fi,

[p-NPP 1 - 2 x 10-B & and 2.7 * 0.3 at [o-NPP] = 2 X 10m3 F&) (3)

During preincubation phosphate

blocks one of the two active

by substrate (4)

with enzyme at pH 4.65 (25' C), inorganic

but does not affect

Phosphorylated

sites

to phosphorylation

enzymic activity.

enzyme is more stable

than native

enzyme in acidic

media.

Exnerimental Materials tally

-- Alkaline

pure from Worthington

saturated

ammonium sulfate

by centrifugation 8.05,

months.

solution.

from a central

All other

metal ions,

(10-6-10-2B

substrate

chromatographi-

as a slurry

buffer

with 0.65 from the slurry (0.05 &, pH

which remained stable

for

phosphate was purchased from Sigma phosphate was prepared by the method

reagents

were "Reagent Grade" materials

by the manufacturer.

The distilled

supply and was shown to be free

of

water was

extraneous

effects

etc.).

Assay Procedure -- Aliquots pH 8.05),

stock solutions

Disodium o-nitrophenyl

and they were used as supplied

(adventitious

Corporation

in N-ethylmorpholine-HCl

to obtain

of Bessey and Love (1962).

was obtained

The enzyme was separated

Disodium p-nitrophenyl

Chemical Company.

obtained

(E. coli)

Biochemicals

and dissolved

u = 0.1 g [NaCl])

several

phosphatase

(10-5-10-3

of stock solutions

of enzyme (10-6-10-5

MJ, and in some experiments

were added to cuvettes

sodium phosphate

(1 cm. path) which contained

310

Ff,

sufficient

Vol. 28, No. 3, 1967

buffer4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to make 2.0 ml of reaction

pH 4.65 with or without substrate

inorganic

to complete the reaction

mixture.

When enzyme was preincubated

phosphate,

reaction

mixture.

In all

was initiated other

at

by adding

experiments

enzyme

was added last. The formation photometer.

of o- or pnitrophenol

was followed

(1 = 355 mn for o-nitrophenol

and 330 my for p-nitrophenol.)

Recording of absorbancy vs. time curves was started initiation (Figure

of reaction.

released

of nitrophenol

within

5-6 seconds after

of the absorbancy vs. time curves

1) to zero time gave absorbancy values

of nitrophenol centration

Extrapolation

with a Cary 15 spectro-

in an "instantaneous

from which the concentration

burst"

was calculated.

The con-

in the burst is assumed to be equal to the concen-

0.162

0.08

40 Time

60

80

100

(Seconds)

phosFig. 1. Alkaline phosphatase catalyzed hydrolysis of o-nitrophenyl phate at pH 4.65. [Enzyme] = 4.55 X lo+ M, [o-NPP] = 5.25 X 10-S 5, temperature = 25.04 * 0.05“ C. 4The buffers were all 0.05 g and were adjusted to an ionic strength of O.lFJ with sodium chloride. Their pH ranges are given below: Potassium Biphthalate - Hydrochloric Acid, pH 3.7-3.9; Acetic Acid - Sodium Acetate, pH 4.1-5.0; - Sodium Hydroxide, pH 5.2-5.7. Potassium Biphthalate

311

Vol. 28, No. 3, 1967

tration

BIOCHEMICAL

of active

activity5

sites

on the enzyme (Hartley

was determined

which remained linear

AND BIOPHYSICAL

to 100X reaction

Results A study of the effect phate on phosphorylation

in nearly

on substrate

cases.

concentration,

phosphatase

in this

of nitrophenol

all

The reaction

section,

burst)

(Figure

pH, and inorganic

(E. coli)

Both concentration

three active

sites

that

obtained

appear to depend

2). 6 The data obtained

with p-NPP can be

tive

studies

its

saturation

Although

the burst

concentrations.

with high concentrations

strong absorption

centration

(2 X 10m3 9

(2.7 f 0.3) and enzymic activity

at lower substrate

is puzzling,

it

increase

Unfortunately,

determined

of absorbancy vs. time curves and S is the substrate

The burst enzymic activity

earlier

increases

corroboraout due to

substrate relationship

conbe-

from the slopes

concentration)

by Heppel et al.

appears to be independent

than

pH 4.65).

with the biphasic velocity

Hownearly

higher

in burst with increasing

is consistent

work (pH ~6) and reported

data require

of p-NPP cannot be carried

tween V and V/S (where V is the reaction

this

phenomenon,

x10-3 M).

is clearly

at 330 mu (E 2 4 X 103r1cm-1,

the continued

of active

and enzymic activity

on the basis of the usual enzyme-substrate

at high 0-NPP concentrations

phos-

by o-NPP and PNPP

The same case can be made for some of the o-NPP data ([o-NPP] ever,

Enzymic

and Discussion

of alkaline

concentration

interpreted

1954).

at pH ~6.

of substrate

is summarized and discussed (magnitude

and Kilby,

from the slopes of the absorbancy vs. time curves

appears to be zero order in substrate

sites

RESEARCH COMMUNICATIONS

found in

(1962) (pH >7).

of pH over the range 3.9 to 5.7 and

smoothly with pH as expected

(Figure

3).

The low

'Enzymic activity is defined as the Slope of Absorbancy vs. Time Curve for NPP Hydrolysis divided by Enzyme Concentration (~-1 sec.-l). 6A good linear relationship between enzyme concentration and burst or enzymic activity was obtained with oNPP (1 X 10m3 MJ and p-NPP (1 X 10m4 @.

312

Vol. 28, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

::h 800 2 $j 600 -4 400

4

2

6

[Substrate]

10

8

X lo4 (MJ

Fig. 2. Effect of substrate concentration on number of active sites ([Nitrophenol] in Burst/IEnzyme]) and enzymic activity (Slope of Absorbancy vs. Time Curve/[Enzyme)). [Enzyme] = 2-10 X low6 kl, pH = 4.68 * 0.05, Temperature = 25.04 * 0.05' C, Substrate: a-o-NPP, A-p-NPP. value

(1.6)

for the number of active

value for the difference It is interesting

that

somewhat from that

sites

extinction

at pH 5.7 is probably

coefficient

(E [o-NPPJ -E [o-nitrophenol]).

the pH dependence of o-NPP hydrolysis

phatase was studied

between phosphate ion and alkaline

by preincubating

in Figures 4 and 5.

lel

one another

active

sites

destruction

In the absence of inorganic

and loss of ensymic activity (Figure

4).

is decreasing of active

1965; Schlesinger,

sites

with increasing

This means that with time,

phos-

the enzyme at pH 4.65 for various

of time in the presence and absence of phosphate ion.

in burst

seems to differ

of p-NPP.

The nature of the interaction

trated

due to a poor

The results phosphate, preincubation

the concentration

are

lengths illus-

the decrease time paral-

of enzymic

a consequence which can be attributed

by acid (Schwartz,

1965).

313

1963; Pigrette

and Milstein,

to

Vol. 28, No. 3, 1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1600

800

2.4 2.0 1.6 4.0

4.4

4.8

5.2

5.6

pH Dependence of Enzymic Activity (Slope of Absorbancy vs . Time Curve/[Enzyme]) and Number of Active Sites ([Nitrophenol] in Burst/[Enzyme]). [Enzyme] - 5 X 10m6 kI, Temperature - 25.04 * 0.05' C, Substrate: q -o-NPP-1.05 x 10-s M, A-p-~~~-2.02 x 1O-4 g.

8

16 Preincubation

24

32

40

Time (Min.)

of Enzyme at pH 4.65 in the Presence and Fig. 4. Effect of Preincubation [Enzyme] = 5 X lO%, Temperature = 25.04 f 0.05' C, Absence of Phosphate Ion. Substrate: p-NPP; 1.05 X low4 JJ. q [HPO,, = ] = 100 [Enzyme] 0 5 X 10B4 M; n [HP04 = I = 0.

314

Vol. 28, No. 3, 1967

h d” R u 2

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

800 700 600

Fig. 5. Effect of Preincubation of Enzyme at pH 4.65 in the Presence and Absence of Phosphate Ion. [Enzyme] = 5 X 10s6 P-l, Temperature = 25.04 * APreincubation 0.05' C, Substrate: p-NPP, 1.05 X 10e4 a. q No Preincubation; Time = 5 min. Preincubation inorganic

of enzyme at pH 4.65 in the presence of a large excess of

phosphate

between the burst proximately

([IWO,, = ]/[Enzyme] and activity

one-half

the reference

somewhat the same fashion Enzymic activity, linearly dently (1963), sites

however, exhibits time.

enzyme against

value of 2 sites

falls

off

difference

rapidly

to ap-

(?) and then decreases in

containing

no phosphate.

no sudden drop but decreases slowly and The presence of inorganic

acid denaturation

as cited

phosphate

earlier

evi-

by Schwartz

but at the same time it causes a decrease in the number of active available

for instantaneous

sistent

with the observations

et aZ.,

(1962);

alkaline

The burst

as in the experiments

with preincubation protects

behavior.

= 100) produces a striking

phosphorylation.

These results

are con-

of Schwartz and Lipmann (1961) and Levinthal

namely, that only one phosphate ion was found attached

phosphatase

in experiments

with P32 labelled

315

inorganic

to

phosphate

Vol. 28, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL

but the enzyme was shown to have two sites brium dialysis

of inorganic

for one preincubation

at [HP04 = ]/[Enzyme] phate the burst

[HPO,, = ]/[Enzyme] = 10.

mixtures

An increase

that

ratio

one-half

in inorganic

by phosphate

tion of phosphate tion

Although

phorylated behavior

at least

in acid media.

phosphate,

a potent

Levinthal,

1960),

hydrolyzed

described

two active

reached a plateau with inorganic

phos-

when enzyme was added

relative

to that

These results

ion is slow compared to

is noteworthy

that

combina-

compared to the acid denaturaenzyme against

above suggest that

sites

acid de-

solution

at the same rate. approximately

If this

phosphos-

aspects of enzymic

is the fact (Torriani,

the enzyme at pH <6.

for by making dephosphorylation process.

several

troublesome

in alkaline

does not inhibit

alkaline

of which one is rapidly

to clarify

Not the least

inhibitor

step in the hydrolysis hydrolyze

increased

4).

in acid media, they fail

be accounted

However, it

phosphate protects

the experiments

phatase contains

it

decrease in burst.

ion with enzyme must be fast

(Figure

was displayed

concentration

of enzyme by phosphate

process because inorganic

naturation

until

The

the same amount of inorganic

phosphate

ester.

way.

The activity

that obtained

which contained

phosphorylation

phosphorylation

5).

ratio

When enzyme was preincubated

was approximately

to reaction

(Figure

of enzyme produced a small but noticeable indicate

in equili-

in another

on [HPO,, = ]/[Enzyme]

time (5 minutes)

with increasing

phosphate.

phosphate

phosphate was illustrated

dependence of burst and activity

directly

for binding

studies.

The effect

sharply

RESEARCH COMMUNICATIONS

that

inorganic

1960; Garen and

This result

cannot

of the enzyme the rate-limiting

were the case, o-NPP and p-NPP should

The data (Figure

2) indicate

that p-NPP is

twice as fast as o-NPP at pH 4.65.

AcknowledPement The author is deeply indebted to Professor F. H. Westheimer for his numerous contributions to the development of this study.

316

Vol. 28, No. 3, 1967

BIOCHEMICAL AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

References Aldridge, W. N., Barman, T. E., and Gutfreund, Ii., Biochem. J., 92, 23C (1964) Bessey, 0. A., and Love, R. H., J. Biol. Chem., 196, 175 (1952). Engstrom, L., Biochim. Biophys. Acta, 52, 49 (1961); 55, 179 (1961); 56, 606 (1962). Engstrom, L., Ark. Kemi, l9, 129 (1962). Garen, A., and Levinthal, C., Biochim. Biophys. Acta, 2, 470 (1960). Hartley, B. S., and Kilby, E. A., Biochem. J., 56, 288 (1954). Heppel, L. A., Harkness, D. R., and Hilmore, R. J., J. Biol. Chem. Levinthal, C., Singer, E. R., and Fetherolf, K., Proc. Nat. Acad. Sci., 48, 1230 (1962). Pigretti, M. M., and Milstein, C., Biochem. J., 94, 106 (1965). Schlesinger, M. J., Subunit Structure of Proteins Biochemical and Genetic Aspects, Brookhaven Symposia in Biology: No. 17 (1964), pp. 70. Schlesinger, M. J., and Barrett, K. J., J. Biol. Chem., 240, 4284 (1965). Schwartz, J. H., Proc. Nat. Acad. Sci., 49, 872 (1963). Schwartz, J. H., and Lipmann, F., Proc. Nat. Acad. Sci., 47, 1996 (1961). Torriani, A., Biochim. Biophys. Acta, 38, 460 (1960).

317