Glycyl-tRNA synthetase: Evidence for two enzyme forms and sigmoidal saturation kinetics

Vol. 70, No. 3, 1976

GLYCYL-tRNA

BIOCHEMICAL

SYNTHETASE:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EVIDENCE FOR TWO ENZYME FORMS AND SIGMOIDAL

SATURATION KINETICS. TERRY A. FRANCIS AND GLENN M. NAGEL Department of Chemistry and the Institute for State University, Fullerton, California 92634

Received

March

Molecular

Biology,

California

30.1976

Summary Two forms of glycyl-tRNA synthetase from E. &have been resolved by DEAE-Sephadex chromatography. The two forms display different kinetic responses to increasing tRNA concentration. One gives a sigmoidal response and a Hill coefficient of 2.2 + 0.2 while the other shows Michaelis-Menten kinetics. It is suggested-that these data may indicate a role for glycyl-tRNA synthetase in the regulation of protein biosynthesis. During DEAE-Sephadex chromatography and ammonium sulfate fractionation the addition of substrates (glycine, ATP, and MgC12) has proven to be of significant value in resolving the two enzyme forms from each other and from contaminating proteins. Each aminoacyl-tRNA synthesis the

by catalyzing

fact

a wide

that

these

diversity

variety

Glycyl-tRNA of 33,000

may be suggestive transcarbamylase (4) yielded studies

or dissimilar

and 80,000

the function was based introduced

(2).

previous

changes

synthetase

bioDespite

they

display

synthetases

as monomers

as well

plots were

and to make purification

two forms

of glycyl-tRNA

have been

distinguished.

synthetase

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

structure

the presence

studies

from

a

as oligomers

for

this described

glycine, with complex

862

such as aspartate synthetase Our

the aim of investigating enzyme.

by Ostrem

with

subunits

ATP, and tRNA.

more efficient.

activity

subunits

of dissimilar

glycyl-tRNA

the possibility

steps

with

to enzymes

with

initiated

to decrease

isolation

Copyright AU rights

Purified

by analogy

comprising

on the procedure designed

reaction,

has an c122g

Although

kinetic

of the subunits initially

to exist

role

Lineweaver-Burk

of glycyl-tRNA

(1).

in protein

aminoacyl-tRNAs,

same basic

from --E. coli

of a regulatory

linear

role

subunits.

daltons

(3),

the

structure

have been shown

synthetase

an essential

of specific

catalyze

in quaternary

similar

plays

the formation enzymes

of sources

of either

synthetase

different

Our isolation

and Berg

(41,

of proteolysis With

these

kinetic

but we during changes,

properties

Vol. 70, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Methods Escherichia coli B cells were purchased from Grain Processing Co, Unfractionated E. coli B tRNA and enzyme grade ammonium sulfate were purchased from Schwartx/kann Inc. GF/C glass fiber filters were obtained from Whatman and [l4C]glycine (90 mCi/mmol) was purchased from California Bionuclear Corporation. Deoxyribonuclease was obtained from Worthington Biochemicals, DEAE-Sephadex A25 as well as phenylmethylsulfonylfluoride were supplied by Sigma Chemical Company. Aminoacylation of tRNA at 37' was monitored by determining incorporation of [14C]glycine into acid-precipitable tRNA following the protocol of Folk and Berg (5). The standard assay solution (0.5 ml) contained 1 mM ATP, 1 mM [14C]glycine (3600 cpm/nmol), 2.62 mg/ml unfractionated tRNA, 40 mM MgC12, 10 mM KCl, 200 ug/ml bovine serum albumin, 4 mM reduced glutathione, and 0.1 M sodium cacodylate (pH 7.1). For saturation experiments, the enzyme preparation was appropriately diluted in 0.1 M cacodylate buffer (pH 7.1) containing, in addition, 30% glycerol to stabilize the enzyme, Reactions were initiated by the addition of one-tenth volume of enzyme solution so that these reaction mixtures contained 3% glycerol. Initial velocities were determined routinely using eight time points. The procedures described below were used in the tRNA synthetase. All manipulations were carried out solid (NH4)2SO4 were made slowly over a period of 30 and the resulting suspension was allowed to stir for tates were separated by centrifugation. Centrifugation performed at 15,000 x g for 45 minutes.

purification of glycylat O-4'. Additions of min with gentle stirring one hour before precipiwas routinely

Twenty grams of E. coli B cell pack was suspended in 20 ml of 0.2 M potassium phosphate buffer (pH 7.0) containing 0.01 M 2-mercaptoethanol, 10% glycerol, 10m4 M phenylmethylsulfonylfluoride and 6000 Kunitz units of deoxyribonuclease. The suspension was sonicated with a Sonifier Cell Disruptor (Model W185 - Heat Systems Inc.) for 6 x 2 minutes so that the temperature remained between 2 and 24O. After centrifugation, the supernatant was diluted to 80 ml with the same buffer and adjusted to pH 7.0 if necessary. The crude extract was made 1.37 M (NH ) SO (35% saturation) with solid ammonium sulfate. After centrifugatlon, .42 t f: e supernatant was made 2.34 M (NH4)2S04 (60% saturation), maintaining pH 7.0. The resulting precipitate was collected and dissolved in 80 ml 0.1 M potassium phosphate, 0.1 M Tris-HCl buffer (pH 8.5) containing 0.01 M 2-mercaptoethanol and 10% glycerol, as well as 5 mM each of ATP, glycine, and MgC12. This solution was made 1.76 M (NH4)2S04 (45% saturation) with solid ammonium sulfate while maintaining pH 8.5. After centrifugation the supernatant was adjusted to pH 7.0 with 2 N HCl containing 1.76 M (NH4)2SO4 and 10% glycerol. The solution was then made 2.26 M (NH4)2SO4 (58% saturation) while maintaining pH 7.0. The suspension was centrifuged and the precipitate dissolved in 10 ml of 5 mM potassium phosphate buffer (pH 7.0) containing 0.01 M 2-mercaptoethanol and 30% glycerol, and dialyzed against 3 x 1 liter of the same buffer. Approximately 70 mg of dialyzed protein was applied to a DEAE-Sephadex column (0.6 x 30 cm) which had been equilibrated with 5 mM potassium phosphate buffer (pH 7.0) containing 0.01 M 2-mercaptoethanol and 30% glycerol. After washing with 100 ml of the same buffer, the column was washed with 0.03 M potassium phosphate buffer (pH 7.0) containing 0.01 M 2-mercaptoethanol and 30% glycerol. The column was eluted with a linear gradient between 150 ml of the 0.03 M potassium phosphate buffer and 150 ml of 0.3 M potassium phosphate (pH 7.0) containing 0.01 M 2-mercaptoethanol and 30% glycerol. Columns Maintaining a 0.2 ml/minute flow rate, 2 ml fractions were collected. were run both in the absence and presence of the substrates ATP, glycine, and measurements of the effluent MgC12 at a concentration of 5 mM. Conductivity were made using an Industrial Instruments Inc. Model RC-16B2 bridge, a63

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1: DEAR-Sephadex chromatography elution profile for fractionation protein, No substrate is present in the buffers, activity (A-A > and conductivity ( ) of collected fractions measured.

solubility Enzyme were

Results Initial

experiments

the crude tion

extract

(35-60%

precipitated

saturation)

experiments

showed

precipitate

over

the enzyme's solubility

identical elution The other results significantly

protein

column are

range

purification

from

fewer

fractions

caused

of ammonium sulfate at elevated

2.

the

concentraSubsequent

enzyme to

concentration,

pH.

Using

in Methods

further,

and that

these

facts,

the

was developed

equal

fractionation

One column

in Figure

fold),

in

and

fold),

the solubility

had 5 mM ATF', glycine,

shown in Figure

of substrates

as described

activity

of ammonium sulfate (1.6

of substrates

columns.

summarized

range

synthetase

purification

(4.1

the effect sample

are

a minimal

procedure

DRAGSephadex data

a wide

was increased

fractionation

To investigate

the glycyl-tRNA

the presence

a narrower

solubility

that over

with

that

gave a much improved

dialized

showed

1.

were

had no substrates

A broad

activity

and MgC12 in all

The activity and was separated

864

portions

eluted into

from

applied

to

added

and the

profile buffers the

can be seen. and the

second

two distinct

of a

column peaks.

in Thus,

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2: DEAK-Sephadex chromatography elution profile for a sample of the same solubility fractionation protein as was used for the elution shown in Figure 1. Present in all buffers are 5 mM ATP, glycfne, and MgC12. Enzyme activity (*A ) and conductivity ( ) of collected fractions were measured.

the presence is

of substrates

interesting

activity

fold

to note,

was not

designated

peak

greater

3.

that

substrates

of the crude

The peak

plots

using

activity

a linear

gave

a bell-shaped of these

data

maintained

plot curve

and

twenty

five-

while

(saturating)

expected

for

Michaelis-Menten

characteristic

plots

865

interaction

in Figure

peak II

linear

Peak II

3.

enzymes

coefficient,

behavior, were

shown in

concentrations,

of many regulatory

the

are

on tKNA concentration.

presented

reciprocal

of the

collected

activity

curve

are also

for

It

half

were

enzymes

data

gave a value

Double

these

of velocity

at standard

Peak I enzyme showed Michaelis-Menten

ATP and glycine.

eluting

fractions

saturation

dependence

of these

resolution.

extract.

measurements

were

strength

Each peak had a specific

a hyperbolic

Eadie-Hofstee

0.2.

the ionic

Peak I enzyme gave a sigmoidal

Invarient

plot

II.

of kinetic

enzyme exhibited

yielded

that

chromatographic

by substrates.

I and peak

than

to increased

however,

shifted

The results Figure

led

however,

while

enzymes.

peak I A Hill

n, of 2.2 f with

respect

and gave Km values

to of

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 3: Kinetic saturation of glycyl-tKt?A synthetase after DF.AE-Sephadex chromatography; enzyme activity from Peak I ( 0-O ) and Peak II ( H ) was determined as a function of tKNA concentration as described in Methods, The data are plotted in the upper panel as normalized initial velocity vs substrate concentration. Eadie-Hofstee plots of these data are shown inThe lower panel; V values were determined from the x-intercepts of the Eadie-Hofstee pl%%,

8 x 10-5 with

M and 1.5 x 10 -4 M for

the values

previously

ATP and glycine

reported

respectively

in good agreement

(4).

Discussion Two

E. coli into

forms

of glycyl-tRNA

extracts.

It

two forms

enzymes

display

is not different

Our purification

synthetase

seems likely an artifact kinetic protocol

that

have not

been

the resolution

of our purification

observed

previously

of the enzyme activity procedure

since

the

properties. which

employed

866

a protease

inhibitor

and elimi-

in

Vol. 70, No. 3, 1976

nated

the

olysis

BIOCHEMICAL

"autolysis"

step

in extracts.

have

observed

leucyl-tRNA between

It

were

substantial

generated

amounts

saturation

kinetics

with

properties

of this

type

not without shown

that

minimum

true

some othermechanism

is responsible

sigmoidal

kinetics

formation

of glycyl-tIU?A

regulation

observed

of protein

The combined glycyl-tRNA

columns.

suggest

for to the

of free

or products. since

activity

in relatively

a mixture

this that

synthetases

Since binding this

it

and that

indicates

kinetics

with

regard

a

as to whether

subunits

seems likely

significance

substrates

or whether

must await that

the

to the

they

may contribute

to the

during

purification

caused

to precipitate

fractionation

enzyme population substrates

--in vivo

use of three

The basis

have

-+ 0.2)

(7).

biosynthesis.

synthetase

ammonium sulfate

here

tRNA and serine

between

basis,

is

has been

a decision

sigmoidal

kinetic

finding

synthetase

however,

the

since

This

(2,2

interactions

the structural

for

shows sigmoidal

finding

limiting

coefficient

for

(6)

of

synthetase

synthetases.

both

sites;

cooperative

we

differences

purification

seryl-tRNA

with

catalytic

prote-

here.

yeast

the Hill

minimize

as has been shown

complete

among the

since

for

Whatever

two enzyme forms

tRNA as a significant

the enzyme displays

study.

the

of glycyl-tRNA

saturation

of two interacting

further

await

one form

however,

we have obtained

should

of the physical

reported

are unusual

sigmoidal

(4)

proteolysis

must

limiting

precedent,

to exhibit

The value

however,

fact

that

determination

of the enzymes

the

and Berg

however,

by limited

Final

two forms,

We regard

used by Ostrem

is possible,

synthetase. these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and to elute

this

effect

complex.

concentrations,

enzyme and enzyme bound aminoacyl-AMP can lead approach

a more narrow

more sharply

is

tightly

to stabilization may prove

useful

as well.

867

Without the

total

to various bound

range

during

from DFAE-Sephadex

may be the conversion

glycyl-AMP high

over

the

of the bulk

addition

of the

of these

enzyme may exist substrates,

intermediates,

to many synthetases

of labile

synthetases,

in the purification

as

and we

of other

BIOCHEMICAL

Vol. 70, No. 3, 1976

The isolation

of two glycyl-tRNA

has been reported capable here

recently

of catalyzing

are

fully

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

active

(8).

synthetase

In this

tRNA aminoacylation. it

appears

unlikely

case,

forms however,

Since that

both

from only

Bacillus

brevis

one enzyme is

enzyme forms

we have observed

reported

an identical

phenomenon, Acknowledgement This investigation was supported by grants from the Brown-Hazen division of Research Corporation and from the Department Associations Council of CSUF. We would like to thank Ms. Phyllis Harley for her aid in preparing the manuscript for publication, References 1. 2. 3. 4. 5. 6. 7. 8.

still, D. and Schimmel, P.R. (1974), Enzymes, 3rd Ed,, 10, 489-538, Ostrem, D.L., and Berg. P. (1970), Proc. Nat. Acad. Sci,, USA 67, 1967-1974, Gerhart, J.C., and Schachman, H.K. (1965), Biochemistry, 4, 1054-10629 Ostrem, D.L., and Berg, P, (1974), Biochemistry, 13, 1338-1348, Folk, W.R. and Berg, P. (1970), J. Bacterial., 102, 139-203. Rouget, P. and Chapeville, C. (1971), Eur. J. Biochem., 23, 452-458. Hertz, H.S., and Zachau, H.G. (1973), Eur. J, Biochem., 37, 203-213, Surguchov, A.P. and Surguchova, I.G. (1975), Eur. J. Biochem., 54, 175-184,

868