Mechanism and binding sites in the ribonuclease reaction II. Kinetic studies on the first step of the reaction

Vol. 7, No. 4, 1962

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NEXXANISMANDBINDING SITES IN TRZ RIBONUCIX~ REACTION II.

KINGTIC STUDIESONTRi3FIRST STRPOF THE RSACTION-' H. Witze12 and S. A. Barnard3

Virus Laboratory,

Received

March

University

of Celifornia,

Berkeley 4, California

26, 1962

It has been suggested that binding to the enzyme through the phosphodiester-monoanion occurs in both the first (RNase) reaction

(1,

2).

However, the velocity

appears to vary very greatly, shown by a qualitative

by following

of the first-step

reaction

depending on the nature of the substrate,

as

comparison of the action of RNaseon different

dinucleoside-phosphates Kinetic

and second steps of the ribonuclease

(1).

information

about the first-step

reaction

the degradation of RNAas substrate.

cannot be obtained

This reaction

is very

complex, since binding is influenced by the macromolecular nature of the substrate and since the observed different

nucleotide

reaction

rate results from cleavage of

diester bonds in RNAwith different

Examination of the degradation of the benzyl or elkyl

velocities

(3).

esters of 3'-cytidylic

acid as smaller substrates using paper chromatographic methods (4, 5, 6) does not have sufficient

accuracy to permit calculations

Wepresent here kinetic measured on several diesters, (CPA,

data for the first particularly

etc. 1, using a spectrophotometric

previous paper (2).

1, 2, 3, 4

of Kmand

values. f4 step of the R&se reaction,

dinucleoside-3',.5'-phosphates method similar

to that used in the

The measurementis based on a spectral change during

See corresponding footnotes to Paper I of this series (2). Abbreviations not defined are as in Paper I. 295

Vol. 7, No. 4, 1962

the reaction,

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

e-go

CpA

cyclic

(ste I> --7Td3+

The absorption at 286 w of 3',5'-CpA and A (a hypochromicity

effect

is the sameas the mixture of 3'-Cp

can be neglected at that wavelength),

absorption of the corresponding mixture of cyclic by the shift

3' - CP + A

Cp + A {g;eiI)+

but the

Cp and A is lower, caused

of the spectrum to shorter wavelength in the cyclic

diesters.

If step I is fast enough, then during I we observe a decrease in absorption followed by en increase during step II

Experimental:

to the initial

values.

Ribonuclease *D" was prepared as in paper I (2).

3'4P

benzyl ester (Cp-benzylyl) was prepared from 3'-Cp and phenyl-diazomethene (7). 3'-Cp methyl ester (Cp-methyl) was a gift phosphates were prepared by hydrolysis hydroxide

(8).

After

of Dr. M. Irie.

of yeast XNA in the presence of bismuth

separation on a Dowex l-X.2

were, where necessary, finally ammonia-water (7:1:2).

Dinucleoside-

purified

ion-exchange column they

by paper chromatography in isopropanol-

They were pure and free of the 2',3'-

isomers, as

shown by complete F&se hydrolysis. The frozen-dried (pH 7.01,

compoundswere dissolved in 0.1 M imidazole buffer

taken to an ionic strength of 0.2 with NaCl.

were determined spectrophotometrically. photometer, as described in paper I,

Their concentrations

In assays in the Cery spectrothe decrease of the absorbance with

time was recorded up to the end-point and, where required,

the subsequent

increase. Initial

velocities

By using the first the reaction i.e.

were determined from normal first-order

part of the reaction

course (and the theoretical

plots. end-point)

could be treated as independent of the second-step reaction,

consecutive reaction

theory was not required.

by the Lineweaver-Burk method. 296

Kmand k3 were obtained

Vol. 7, No. 4, 1962

Results:

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AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

Table I shows the results. TABLE I

Kinetic

constants at pi 7.0 (imidaeole buffer), ‘Km

Substrate

in

I4 x

26O

10 -3

Substrate

Km

k3

CPA

3000

1.0

UPA

CPG

500

3.0

UPG

CPC

240

4.0

UPC

40

CPU

27

3.7

UPU

11

mm

--

300 3.7 I

Cp (cyclic)

5.5

3.3

Cp-benzyl

2

3

@-methyl

0.5 (from initial

velocity,

With Cp-methyl the rate of the first

step is so slow compared with

in deriving

velocities

state was reached after theoretical therefore

'The half-time reaction

portion

(1 minute) could be used

The fastest

decrease and subsequent return: formed as obligatory

shows a decrease in the

for this substrate.

85% decrease.

With UpU the steady

substrates showed the

the cyclic

phosphates are

intermediates.

of RNasehydrolysis

of cyclic

Cp formed during the

of CpA was found to be the sameas in the reaction

alone, or of cyclic Discussion:

Cp-benzyl,

decrease, reaching a pseudo steady

only the very initial

the initial

was possible.

step and therefore

absorbance to only 285; of the theoretical state near there;

5. 0

assuming Km in the samerange)

the second step that no reasonable calculation also, is slower in the first

2.2 1

Up (cyclic)

of cyclic

Cp

Cpin equimolar mixture with adenosine.

The Km values for the first-step

substrates do not differ

from

the values for the second-step substrates;

only in the cases of CpA and UpA

is

An interpretation

Km

a little,

but significantly,

lower. 297

of these data

BIOCHEMICAL

Vol. 7, No. 4, 1962

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

in terms of a commonbinding site in the enzyme and also in the substrates will

be given elsewhere in connection with further

dence of the kinetic In contrast,

constants and on inhibition

in both steps.

k5 shows enormous differences,

of tne second alcohol and particularly difference

results on the pH depen-

dependent on the nature

of the second nucleoside.

between the benzyl and methyl esters Ithe isopropyl

even more slowly than tne methyl ester electrophilicity

at the phosphorus

tendency" of the alcohol group. value for the cyclic the nucleophilicity molecule.

diester,

ester reacts

[5]) csn be explained by the decreased corresponding to the "leaving

atom,

The samereason may explain the higher

although here additional

and position

The

of the attacking

In the dinucleoside-phosphates

differences

exist in

2'-OH group and the "20

the differences

cannot be explained

on the samebasis, because the ester bond to the phosphorus is in all Therefore,

the same5'-OH group of the ribose. second factor philicity

we have to assumethat the

in the catalysis,

of the pyrimidine base (2), is itself

nucleoside. is

determining the velocity

cases

namely the nucleo-

influenced by the second


- the reaction

rate

6ooo-fold higher in CpA than in Cp-methyl under the conditions mentioned -

can be explained only on the basis of a <-interaction

between the pyrimidine

base and the second base, increasing the polarisability

and therefore

nucleophilicity reaction

of the catalysing

(1, 2) this is sterically The interpretation

system.

In the position

in the terms of fl-interactions,and

(perhaps through the C-6-substituents),is

facts:

the purines activate

have a lower degree of sromaticity,

more strongly especially

of the rates for cyclic

Cp and cyclic

not a localised

based on the following

than the pyrimidines, uridine;

cannot be explained by the known H-bonding affinities The ratio

required for

possible, as can be demonstrated on models.

interaction (I)

the

which

(2) the differences of the pairs concerned.

Up (2.5-5,) is the sameas

that for CpA and UpA or for CpU and UpU, although in UpA an activation

should

occur by an H-bridge withdrawing the proton from the C-6-OH group, and in CpUan inhibition

by the transfer

of a proton to the C-&NH2 group. 298

Thus

Vol. 7, No. 4, 1962

BIOCHEMICAL

the contribution

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of the second base to the nucleophilicity

of the pyrimidine

base involved seemsto be independent of the C-6-substituent. greater - 6-fold invalidate

- difference

has

now

between CpC and UpC is not sufficient

this argument); (3) a mixture of cyclic

show acceleration

(i.e.

Cp and adenosine does not

adenylyl-(3',5*)-cytidine

(4) there is evidencetdatahypochromicity

side-phosphates is based on a similar

to

although such an acceleration

of the second step reaction,

been observed in ApGcyclic-p

phosphate);

(The rather

effect

s-interaction

2',3'-

in the dinucleo-

between the two bases,

according to Michelson (9), and in general (although all cases have not been tested) the sameseries is usually shokm for the influence base:

A > G) C) U (9,

IO).

This interpretation polarisability proposed (I,

d-interactions,

centre,

increasing the

requires a mechanismof the type

In such a case, catalysis

increase in polarisation bility

in terms of

at the nucleophilic 2).

of the second

does not arise from a permanent

but from an increase of the amplitude of polarisa-

in the C-2'-OH bona, by joining

the hydrogen to a highly resonating

system, which can be amplified by other appropriate and more examples of this type of catalysis

Liebids Ann.Chem., a,

will

systems.

A discussion

be given later.

(II

1Jitze1, il.,

(2)

Uitzel,

(3)

Rushizky, G.';i., Knight, C.A. and Sober, H.A., J.Biol.Chem.,

H. and Barnard, &A.,

191 (1960)

Biochem.Biophys.Res.Comm. (previous paper) 2&

2732

(1961) (4)

Davis, F.F. and Allen,

(5)

Barker, rJ.R., ilontague, M.D., MOSS, R.J. and Parsons, M.A., J.Chem.Soc., (London), 3786 (1957)

(6)

Hummel,J.P.,

(7)

&own, D.X. ana Todd, A.R.,

J.Chem.Scc.(London), 2040 (1953)

(8)

Dimroth, K. and Titzel,

Liebigs Ann.Chem., 620, 109 (1954)

(9)

Michelson, A.M., Ann.Rev.Biochem., 0,

(IO)

Michelson, A.M., J.chem.soc.(London),

Flares,

F.W., Z.Biol.Chem.,

M. and Nelson, C.A.,

Ii.,

299

217, 13 (1955)

J.Biol.Chem.,

133 (1961) 3655

(1959)

9,

717 (1958)