The structure of the metal-nucleotide complex substrate for yeast hexokinase

Vol. 57, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE STRUCTURE OF THE METAL-NUCLEOTIDE

COMPLEX SUBSTRATE FOR YEAST HEXOKINASE

Cecil Cooper Department of Biochemistry Case Western Reserve University Cleveland, Ohio 44106

Received

January

24,1974 SUMMARY

A comparison has been made of the kinetic parameters obtained with yeast hexokinase using Mg2+ or Ni2+ as metal ion activator and ATP or tubercidin-5'triphosphate as nucleotide substrate. It is concluded that the relative specificity of the enzyme for MgATP 2- does not involve a contribution arising from an interaction of the metal ion with the purine ring of the nucleotide.

It tide

has been proposed,

substrate

catalyze other

the

2+

, the

occurring

for only

of the

primarily

depending to complex

on the with

aqueous

8 and y phosphates

phosphates (Mg

2+

, Ca

used.

G 1974 by Academic Press, Inc. of reproduction in any form reserved.

about

of ATP (Mn

2-t , Zn2+)

434 Copyright Ail rigl1t.r

the requirement complex

is

(11-13).

of a metalthe

recognition

A second

possibility

essential.

between

The

these

two possi-

of metal-

a good deal

of evidence,

of possible

2+

(8-10).

the structure

react

metal , co

with

both

2+ , Ni2+)

More

the

adenine

interactions ions

recently,

and

naturally

in which

may provide

is

ions

addition

donor

to choose

Divalent

in

as the phosphate

catalysis.

A range

metal

Both

ring

cations

also

and Ca2+ (5,7).

of the

There

divalent

ion

is

is known

of ATP.

metal

three

below

the nucleo-

enzyme will

shown that,

for

for

that

divalent

to the phosphates

solution.

that

groups

specific all

metal

of what

from nmr studies,

and the phosphate

areas

described

in

has been

the purine

reactive

advantage

It

explanations

with

This

when other

The structure

interacts

the binding

(l-4).

of ATP can serve

two obvious

and/or

complexes

MgATP 2-

information,

by Mn2+ (5,6)

stimulated analogs

metal

binding

of kinetic

are used.

as substrate.

by taking

nucleotide

the

is

of the experiments

bilities

ring

is

at least

bound

features

purpose

reaction

complex

phosphate

hexokinase

triphosphates

are

nucleotide

basis

of glucose-6-phosphate

and synthetic

There

that

yeast

formation

nucleoside

toMg

is

for

on the

exist

have been or with

reported

only

phosphorus

Vol. 57, No. 2, 1974

Fourier

transform

three via

BIOCHEMICAL

analysis

phosphates

the N-l

not

water

molecule

Ca2+,

reportedly

or N-3 positions

The rationale obtained If

with

the only

is'the interact

two different

then

lent

metal

complex action 2+

be too

interaction

is

complex

ATP at N-l

We have previously

found If

(15).

should

(17),

reported

results.

If,

ring

(14)

that

ring

complex

hexokinase

(Type XV) were

purchased

The preparation was a previously for

the value

(Type

calculations of 50,000

C-300)

Since

different. forms

almost

similar

should equivalent

activating

metal

AND METHODS

and glucose-6-phosphate

dehydrogenase

from Sigma.

of the enzymes

and reagents,

and the performance

described

(10).

The stability

was 48,000

(18).

This

obtained

by correcting

435

is

constant

in excellent

the data

of Frey

of

inter-

two complexes

the

as diva-

in place

the metal-ring

TuTP is

ATP

in the Ni-TuTP

and probably

these

is

we selected

that

be quite

when Mg2+ is

hexokinase

ions

an N-l/N-3

7 of the

and N-3

of view

metal

the purine

comparison

ions.

and Mg2+ and Ni2+

we have

or N-3

point

on the

with

parameters

(10).

Yeast

used

with

metal

different

ion with

atom in position

at N-l

divalent

that

as nucleotides

and Ni-TuTP

yeast

the kinetic

have no bearing

case of MgATP and Mg-TuTP

for

fact

to make the

(TuTP)

MATERIALS

assay

all

2+ interact

manner

from the enzymatic

sphere

In order

with

to ATP as a substrate ion

with

of ATP (15,16),

unspecified

to compare

of the metal

Ni-ATP

to interact

is

the

From nmr studies

then

different.

react Mn

ring

and two different

an N-7 outer

interaction

in the

adenine

an as yet

below

should

TuTP has a carbon

important

in

then

ring

differences.

ring

appears

complexes

the

in adenine.

the is

the

ions.

2+ and Ca2+ also

and Co2+ and possibly

of the complex

-5'-triphosphate

the nitrogen

Mg

feature

comparing

show large

and tubercidin

interacts

nucleotides

with

hand,

2+

Mg

the N-7 of the

interaction,

differently

important,

with

Ni

of the experiments

important

on the other

While

that

RESEARCH COMMUNICATIONS

(14).

metal-phosphate

might

has revealed

of ATP (14).

a bridging

2+ , but w

AND BIOPHYSICAL

for

of the 2MgATP

agreement

with

and Stuehr

(19)

not

Vol. 57, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 K Nucleotide

Metal

V mw (pnoles-min

Ion

ATP

% 2+

0.20

+ -03

ATP

Ni2+

0.16

+ .Ol

TuTP

w

0.23

+ .03

TuTP

Ni

0.11

+ .Ol

2+ 2+

-1 .mg-l)

356 + 13 Sl+

2

306 + 18 80f

3

The assay medium contained 50 mM HEPES (N-2-hydroxyethyl piperasine-N'2-ethane sulfonic acid) neutralized with tetramethylammonium hydroxide, 50 mM tetramethylammonium nitrate, 9 mM glucose, 0.4 mM NADP, 3.2 units glucose-6-phosphate dehydrogenase and triple distilled water to a final volume of 320 ~1 and a final pH of 8.0. After a 5 min preincubation at 25' the reaction was started by the addition of a 5 ~1 aliquot containing 0.05 unit yeast hexokinase. For each analysis 9 concentrations of metal nucleotide ranging from 0.08-0.9 mM were used. The values given are the mean f S.D.

for

temperature

by using value

and potassium

the van't

for

the binding

as 30 (19).

was obtained

ture

and potassium

equation

there

by correcting binding.

are stronger was also

was used

for

the value

nitrogen

no interaction

with

stability

constant

and Stuehr

a copper

containing

between

Ni

2+

ligands

while

of 0.1

(19)

specific

2+ Cu and the HEPES buffer.

betwen with

the

was made

AH (20)

strength

of Frey

Experiments

correction

of 2600 for

of KATP at an ionic

of 225,000

was no interaction

actions

and a value

constant

A value

This

there

Hoff

The temperature

binding.

the

M was taken 2of NiATP . for

tempera-

electrode

Since

than

Ni

is

that

2+

showed

Cu2+ interit

was assumed

and HEPES.

RESULTS AND DISCUSSION The results kinetic

are shown

parameters

result

nucleotide

combination.

free

ion

metal

experiments far

as the enzymatic

with

the phosphate

from

1.

from

groups

is

and not

in metal were

5-150

the important reaction

The main

changes

The results

was varied

is that

in Table

and not when the

The conclusion

alterations

from

the

436

is ring.

the

drawn

interaction It

in

the metal

concentration

of the metal-nucleotide

concerned with

ion

identical

polar.

feature

point

is possible

of

from

these

complex of the metal that

ion-

the

as ion inter-

Vol. 57,No.2,1974

action

of the

BIOCHEMICAL

phosphate

bound

on the enzyme-substrate of the nucleotide of the with

ring

potential

sites

with

is known

on the

ring

complex

to undergo

with

the nucleotide

the enzyme alters

in the hardness-softness

which

ion

Such a situation

complex.

the metal-phosphate

volved

metal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

their

is

during

if

ability

One potentially

property variation

arise

is

restricted

an interaction

the hardness-softness

and therefore (17).

could

ring

to coordinate

important

the microscopic the course

properties

parameter

dielectric

in-

constant

of many enzymatic

reac-

tions. ACKNOWLEDGMENTS This Science is

work

was supported

Foundation.

gratefully

by a research

The expert

technical

grant assistance

(GB-12747)

from the

of Ms. Ksenja

National

Dimitrov

acknowledged. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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