A phosphoribosyl-enzyme covalent intermediate in the first enzyme of histidine biosynthesis

BIOCHEMICAL

Vol. 38, No. 4, 1970

A

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHOSPHORIBOSYL-ENZYME COVALENTINTERMEDIATE

IN THE FIRST ENZYMEOF HISTIDINE BIOSYNTHESIS Robert M. Bell and D. E. Koshland, Jr, Department of Biochemistry, University of California Berkeley, California 9472C

Received November 25, 1969 SUMMARY : Evidence is presented that the mechanismof phosphoribosyl-adenosine triphosphate: pyrophosphate phosphoribosyltransferase (the first enzyme of histidine biosynthesis) proceeds through a covalent phosphoribosyl-enzyme intermediate. The intermediate has been demonstrated after incubating the enzyme with 14C-PRPPunder both "native" and denaturing conditions. The intermediate also forms from the reverse direction as demonstrated by an initial burst phenomenonwhen the enzyme is mixed with its product, PR-ATP. Thus, exchange data coupled with specificity requirements appears to provide sound evidence for a covalent enzyme-substrate intermediate. The first

enzyme of the histidine

typhimuriwn is of particular regulation

interest

of the pathway (l-3)

sugar-protein

intermediate

adenosine triphosphate:

+

pathway of Salmonella

because it plays an important role in

and because its mechanismof action suggests a Martin found that the enzyme (phosphoribosyl-

pyrophosphate phosphoribosyltransferase

which catalyzes the overall

PRPP

(4).

biosynthetic

(E.C. 4.2.1C)

reaction shown in Equation 1 also catalyzes

PR-ATP

ATP

1 PRPP, 5-phosphoribosyl-1-pyrophosphate; Abbreviations: phosphoribosyl)-adenosine triphosphate. 539

+

an

PPi

PR-ATP, N-1-(5'-

Vol. 38, No. 4, 1970

BIOCHEMICAL

exchange between

32

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P-pyrophosphate and PRPP' in the absence of ATP and between

14C-ATP and PR-ATP in the absence of PRPP(4). valent intermediate roff,

The first

postulation

by such exchange reactions was the classic study of Doudo-

Barker and Hassid in the case of sucrose phosphorylase (5).

exchange per se is not proof of an enzyme-substrate intermediate been pointed out that exchange data coupled with specificity should be evident for a covalent intermediate valent intermediate has been verified

predict

(7,8).

Although (6), it has

requirements

The prediction

of a co-

in the case of sucrose phosphorylase by The sameexchange-specificity

the studies of Abeles and Voet (9,lO). would therefore

of a co-

criteria

that a covalent intermediate should be found in the

phosphoribosyl-adenosine triphosphate:

pyrophosphate phosphoribosyltransferase

enzyme. In this paper we report evidence for such an intermediate. MATERIALSANDMETHODS Phosphoribosyl-adenosine triphosphate:

EnzymeAssay. phoribosyltransferase

activity

as modified by Voll,

was assayed by the methods of Ameset al.

modification

was purified

of the procedure of Voll et al.

ed in three different

buffer

pyrophosphate phospho-

from Salmonella typhimuriwn strain El1 by a

Sephadex G25 chromatography.

(11).

G-25 Sephadex chromatography was perform-

systems: Buffer A - 0.01 M Tris-HCl,

0.5 mMEDTA, 1.0 mMdithiothreitol, L-histidine;

0.10 M NaCl,

pH 7.5; Buffer B - Buffer A plus 0.4 mM

and Buffer C - 0.01 M potassium phosphate and 5

I%-PRPP.

(1)

Apella and Martin (11).

Enzyme. Phosphoribosyl-adenosine triphosphate: ribosyltransferase

pyrophosphate phos-

l4 C-PRPPwas synthesized enzymatically

phosphate (Schwarz Bio-Research, Inc.)

urea, pH 7.0. 14 from C-ribose-5M

and ATP using PRPPsynthetase from

SalmonelZa typhimurium (12) and purified

according to the procedure of Korn-

berg and Khorana (13). N-I-fS’-phosphoribosyI)-ATP. modification

PR-ATPwas prepared enzymatically

of the procedure reported by Smith and Ames (14).

540

by a

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS In an attempt to demonstrate the existence of an intermediate was incubated with l4 C-PRPPand magnesiumchloride. phatase was added to displace the equilibrium 14C-PRPP+ E Mg++,

Yeast inorganic pyrophos-

in favor of formation of the

14C-PR-E + PPi I PPiase

I 4

intermediate

as shown in Equation 2.

the enzyme

2Pi

Urea was then added until

tion reached 6 M where the enzyme is known to be inactive

the concentra-

and dissociated

This solution was placed on a G-25 Sephadex column equilibrated

(11).

with 5 M urea.

The results shown in Figure 1 present strong evidence for a covalent inter-

10

0 FRACTION

20

NUMBER

Figure 1. Resolution of 14C-phosphoribosyl-enzyme intermediate from 14c14C-PRPPusing G-25 Sephadex chromatography under denaturing conditions. Phosphoribosyl-enzyme complex was produced in a reaction mixture containing 1.4 mg protein in 0.45 ml buffer A, 4.5 umoles MgC12, 0.5 umoles 14C-PRPPand 1.8 units of yeast inorganic pyrophosphatase (Worthington). After 30 seconds at room temperature, solid urea was added to make the solution 6 M and the mixture was applied to a G-25 Sephadex column (1.5 x 25 cm) equilibrated with The control consisted of enzyme buffer C and calibrated with Blue Dextran. iHj. already in 6 M urea before the 14C-PRPPwas added. Radioactivity, control radioactivity, O-O. 541

Vol. 38, No. 4, 1970

A radioactive

mediate. experiment

whereas

PRPP was added Since possible

without

it

in this

could

in a control

enzyme already

the intermediate

an experiment

2) that

in the breakthrough

the peak was absent

to inactive

denaturation

Figure

peak was found

the enzyme does not

that

therefore

which

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

water

after

in which

incubation

experiment

be argued

that

also

with

might

4

denaturation

6

FRACTION

12

it

seemed and

the enzyme was chromatographed

in

Again

it

was noted

the breakthrough

the enzymatic

arise

from

activity.

non-covalent

(cf.

volume Although binding,

s’

Radioactivity-/

0

without

appeared

coincided

such a peak

acceptor,

14c-PRPP.

with

a peak of radioactivity

above 14c-

in which

as a normal

be isolated

was initiated

experiment

in the

in 6 M urea.

utilize might

volume

16

20

24’

NUMBER

Figure 2. Resolution of l4 C-phosphoribosyl-enzyme intermediate from 14C-PRPP using G-25 Sephadex chromatography under non-denaturing conditions. Phosphoribosyl-enzyme complex was produced in a reaction mixture containing 1 ml of enzyme in buffer A, 0.15 umoles MgC12, 0.5 umoles 14C-PRPP and 2.7 units of yeast inorganic pyrophosphatase. After two minutes at room temperature, 50 ul of a 15 nM L-histidine solution at pH 8.5 was added. l’he reaction mixture was applied to a G-25 Sephadex column (1.5 x 30 cm) equilibrated with buffer B at 4’C. The control did not contain enzyme. O-O, enzyme activity; A-A, radioactivity, Eta, control radioactivity.

542

Vol. 38, No. 4,197O

this alternative

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was eliminated by the finding

that the peak was absent when

either pyrophosphate or ATP was added to the labeled enzyme. The absence of a peak after

such treatment is consistent with removal of a covalently

bound

14C-phosphoribosyl moiety from the enzyme by reaction with these substrates. Further evidence for the existence of a phosphoribosyl-enzyme intermediate was obtained by difference the enzyme. The high extinction phoribosyl-glycosidic

specta at 290 mu when PR-ATPwas added to at 290 mu is associated with the N-l’-phos-

bond of PR-ATP (1).

As shown in Figure 3, a

absorption at 290 mu was observed which was proportional centration. glycosidic

loss

of

to the enzyme con-

This is consistent with the breaking of the N-l’-phosphoribosylbond of PR-ATP and the formation of a phosphoribosyl-enzyme.

is characteristic

of an initial

It

burst phenomenonsuch as that observed for

chymotrypsin in the presence of various substrates (15-17).

ENZYME CONCENTRATION

(M x 106)

Figure 3. Formation of phosphoribosyl-enzyme intermediate from PR-ATP. Difference spectra at 290 mu obtained using tandom cells. Loss of PR-ATPwas calculated from the observed decrease in ‘absorption at 290 mu from the difference in the extinction coefficients between ATP and PR-ATP at 290 mu, pH 8.3 (18). PR-ATP at 6.86 x 10V4 M was madeup in 1 M Tris buffer, pH 8.5. Enzyme at 9.2 x loo6 M was diluted in 1 M Tris, pH 8.5, to the &sired concentration. Both the PR-ATPsolution and the enzyme solution were incubated with 4.5 units of yeast inorganic pyrophosphatase for 20 minutes at room temperature to eliminate traces of pyrophosphate. 543

Vol. 38, No. 4, 1970

Finally, per mole PRPP .

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

stoichiometries

of enzyme have been

Since

the

is

consistent

with

The finding

of a covalent

it

a tool

provides

and the nature

of the

for

The essence

of this

the donor

and Y the

exchanges

of the

displacement

possibility

the

shown schematically

when X* can occupy

(ATP cannot

occupied occupy

by X.

the PP site

effector,

4.

reaction,

Y* can only

If

for

B-X is

we can see that

occur

normally

If the

support

intermediate.

in Figure

the site

interesting

adds further

transfer

(ll),

structure.

of a covalent

X* and B-Y with

of the i4C-

of the allosteric

also

bound

subunits

enzyme is

role

as evidence

in an enzymatic

normally

this

It

acceptor

activity

subunit

the

intermediate.

B-X with

specific

groups

of 6 identical

in

is

the site

the

reported

argument

type

of phosphoribosyl

to consist

investigating

criterion

mechanism

Y* can occupy

from

intermediate

covalent

the exchange-specificity

this

calculated

enzyme has been found

the stoichiometry

since

up to 6.0 moles

by a single

occupied

specificity

by Y or pattern

and v,ice versa),

then

EXCHANGE CRITERIA FOR TRANSFER REACTION OF TYPE B-X + Y -

excludes

positive

B-Y + X

A. SINGLE DISPLACEMENT MECHANISM

EXCHANGE

(B.X + X’

-

B-X’

+ X) OCCURS

ONLY IF X* CAN OCCUPY

x...B-x* z &f=y&

n X-B x*

Y SITE

= Yg$jf x B-x*

‘6. DOUBLE DISPLACEMENT MECHANISM gJ$y&=&=~ EXCHANGE

Figure

4.

(B-X’

Illustration

+ X* -

B-X’

+ Xl OCCURS

EVEN

IF X* CANNOT

of the exchange-specificity

544

OCCUPY

criterion

y BITE

(see

text).

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

exchange experiments indicate

a double displacement mechanism. Whenthis criterion

was formulated on theoretical

grounds, no covalent intermediates had been

isolated.

Today it appears that whenever the exchange-specificity

criterion

have

been met that an appropriate search has yielded the expected covalent intermediate. It

would therefore

an indicator

appear that the exchange-specificity

of an intermediate.

of stereochemistry

can be verified

criterion

is sound as

It remains to be seen whether the prediction in this case as it has been for sucrose

phosphorylase. Ackndedgwnent

.

This work has been supported by a research grant

(AM-GM-09765) from the National Institutes has been aided by a training

of Health and one of US (R.M.B.)

grant (5 TO1 GM 31).

We gratefully

acknowledge

valuable discussion with Professor Bruce Ames and Dr. Harvey Whitfield.

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18.

Ames, B. N., Martin, R. G., and Garry, B. J., J. Biol. Chem., 236, 2019 (1961). Ames, B. N., Goldberger, R. F., Hartman, P. E., Martin, R. G., and Roth, J. R., in Koningsberger and Bosh (Editors), Regulation of Nucleic Acid and Protein Biosynthesis, Elsevier Publishing Co., Amsterdam, 1967, p. 272. Kovach, J. S., Phang, J. M., Ference, M., and Goldberger, R. F., Proc. Natl. Acad. Sci. U.S., 63, 481 (1969). Martin, R. G., J. Biol. Chem., 238, 257 (1963). Doudoroff, M., Barker, H. A., and Hassid, W. Z., 3. Biol. Chem., 168, 725 (1947). Boyer, P. D., Ann. Rev. Biochem., 29, 15 (1960). Koshland, D. E., Jr., Biol. Rev. Cambridge Phil. Sot., 28, 416 (1953). Koshland, D. E., Jr., Discussions Faraday Sot., 20, 142 (1956). Voet, J., and Abeles, R. H., J. Biol. Chem., 241, 2731 (1966). Voet, J., and Abeles, R. H., Federation Proc., 28, 602 (1969). Voll, M, J., Appella, E., andMartin, R. G., J.xol. Chem., 242, 1760 (1967). Switzer, R. L., J. Biol. Chem., 244, 2854 (1969). Kornberg, A., and Khorana, H. B.,ochem. Prep., 5, 110 (1961). Smith, D., and Ames, B. N., J. Biol. Chem., 2, 1848 (1964). Hartley, B. S., and Kilby, B. A., Biochem. J., E, 288 (1954). Kezdy, F. J., Clement, G. E., and Bender, M. L., J. Am. Chem. Sot., s, 3696 (1964). Bernhard, S. A., and Gutfreund, H., Proc. Natl. Acad. Sci. U.S., 53, 1238 (1965). Smith, D., and Ames, B. N., J. Biol. Chem., 240, 3056 (1965).

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