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PROTEINS: Structure, Function, and Genetics 24:370-378 (1996)
Experimental and Theoretical Study of Electrostatic
Effects on the Isoelectric pH and the pK, of
the Catalytic Residue His-102 of the
Recombinant Ribonuclease From Bacillus
amyloliquefaciens (Barnase)
Katrin Bastyns,' Matheus Froeyen,' Jose Fernando Diaz,' Guido Volckaert? and Yves Engelborghs'
'Laboratory of Chemical and Biological Dynamics and 'Laboratory of Gene Technology, University of Leuuen,
B-3001 Leuuen, Belgium
ABSTRACT
Barnase, the guanine specific
ribonuclease of Bacillus amyloliquefaciens,
was subjected to mutations in order to alter the
electrostatic properties of the enzyme.
Ser-85 was mutated into Glu with the goal to
introduce an extra charge in the neighborhood
of His-102. A double mutation (Serd5Glu and
Asp-86-Asn)was introduced with the same purpose but without altering the global charge of
the enzyme. A similar set of mutations was
made using Asp at position 85. For all mutants
the PI was determined using the technique of
isoelectric focusing and calculated on the basis
of the Tanford-Kirkwood theory.
When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the
Ser-85-Aspmutant, the experimental PI drop is
bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the
compensation is not achieved.
The effect of the mutations on the pK, of His102 can be determined from the pH dependence
of the k,,,/K,
for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the the method of Honig. In this case
the agreement is very good for the Glu-mutants
and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Aspmutants is, however, the same as the wild type,
as shown by stability studies using urea denaturation. Molecular dynamics calculations,
however, show that in the double Asp-mutant
His-102 (H+) swings out of its pocket to make a
hydrogen bridge with Gln-104 which should
cause an additional pK, rise.
The effect of the Glu-mutations was also
tested on all the kinetic parameters for GpC
and the cyclic intermediate G > p at pH 6.5, for
RNA at pH 8.0, and for poly(A) at pH 6.2. The
effect of the mutations is rather limited for the
dinucleotide and the cyclic intermediate, but a
strong increase of the K, is observed in the
0 1996 WILEY-LISS, INC.
case of the single mutant (extra negative
charge) with polymeric substrates.
These results indicate that the extra negative
charge has a strong destabilizing effect on the
binding of the polymeric substrates in the
ground state and the transition state complex.
A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R.,
Biochemistry 3316461653,1994) shows that the
extra negative charge points towards the P2
site. 0 1996 Wiley-Liss, Inc.
Key words: barnase, site-specific mutagenesis,
RNAse, RNA, poly(A), pH-profile,
enzyme kinetics, electrostatics,
Delphi, Tanford-Kirkwood
INTRODUCTION
Barnase is a guanine-specific endoribonuclease
from B . amyloliquefaciens. The crystal structure has
been solved.' The mechanism of action consists of
two steps: in a first step of transesterification, a cyclic intermediate is formed, which is hydrolysed in a
second step. His-102 acts as a general acid, and
Glu-73 as a general
Like many enzymes that use polymeric substrates, barnase hydrolyses RNA much more efficiently than dinucleotides, indicating the presence
of multiple subsites on the enzyme, and the influence of the interactions at these subsites on the conformation at the primary site.
A detailed study with oligonucleotides of the type
Zp,Gp,Xp,Y was made (Day et al., 1992) and this
study revealed that: (1) the minimal substrate is
GplX (kcat/KM lo4 M-ls-'); (2) a considerable improvement is obtainedhhen pz is present (kcat/KM
lo6 M-ls-' 1; (3) the limit of diffusion control is ob-
-
-
Received May 5, 1995; revision accepted October 3, 1995.
Address reprint requests to Yves Engelborghs, Laboratory of
Chemical and Biological Dynamics, Celestijnenlaan 200 D,
B-3001 Leuven, Belgium.
371
ELECTROSTATIC EFFECTS IN BARNASE
Fig. 1. Stereographic view of the ribbon representation of the main chain of barnase showing
the position of the three important residues: Ser-85, Asp-86, and His-102. (The drawing was
generated using the program MOLSCRIPr').
-
tained when Y is also present (kcat/K,
los
M-ls-'); (4) Zp, does not influence the kinetic parameters. Furthermore it is remarkable that the pH
value of maximal kCat/K, is a t a much higher pH for
RNA hydrolysis than for the hydrolysis of dinucle~tides.~
In our previous study6 we mutated Glu-60 into
Gln and proved that this mutation was deleterious
for the activity of barnase on dinucleotides but not
on polymeric substrates, indicating a different conformation of the substrate in the binding site when
dinucleotide and polymeric substrates are compared.
In this article, mutations are made with the goal
to introduce a n extra charge in the neighborhood of
the catalytically important His-102. This goal can
be achieved by replacing Ser-85 by Glu or Asp. In
the case of the Ser-85-Glu mutant, the COO- is at a
distance of 5 A form the His-l02(Ne3) and 11A from
the negative charge of the Pi of the dinucleotide
GpG (own modelling). The position of the three residues considered is shown in Figure 1. This operation can also be achieved by the simultaneous mutation of Asp-86 into an Asn, with the goal to keep
the total charge of the protein constant. The negative charge of Asp-86 is at 9 A from His-102 ( N E ~ ) .
The results show that this is very important for
polymeric substrates at pH 8.0 and 6.2, but much
less for dinucleotides and for the cyclic intermediate
at pH 6.5.
A study of long range (between 12 and 24 A) surface charge-charge interactions on the His-18 of
barnase was performed by Loewenthal et a1.l' showing that a good agreement was obtained between
calculated and experimentally determined ApK,
values, using the DELPHI program. The experimental ApK, values were obtained from fluorescence titrations, where the fluorescence of Trp-94 is
strongly quenched by Hi~-18.~-'
In this study we concentrate on the electrostatic
effects at the catalytically important His-102 residue, and we want to discriminate between local and
global charge effects, and between dinucleotide and
polymeric substrates.
MATERIALS AND METHODS
Restriction enzymes were purchased from Boehringer Mannheim (Mannheim, Germany). Dideoxy
sequencing was done with the T, sequencing kit
from Pharmacia (Uppsala, Sweden). SP-Trisacryl
was purchased from IBF Villeneuve (La Garenne,
France) and the Mono S and Phenyl Superose column were purchased from Pharmacia. The dialysis
tubing (cut off 3,500 and 13,000), was obtained from
Spectrum Medical Industries (Los Angeles, CA). The
purified samples were concentrated with Centricon
microconcentrators (Amicon, Danvers, MA). The
substrates GpA, GpC, ApA, and RNA were obtained
from Sigma Chemical Co. (Dorset, UK). Poly(A) was
obtained from Pharmacia. Reagent-grade buffer materials and distilled and Millipore filtered (0.45 pm)
water were used in the preparation of buffers.
The bacterial E . coli strains used in this study
were WK6 (A(lac-proAB) galE strA [F'lucP ZAM15
proA+B+]) and WK6 mutS (A(1ac-proAB)gulE strA
rnutSl25::TnlO [Flaclq ZAM15 proA B I). The
WK6 strain was used for production of barnase,
while the mutS derivative served for the mutagenesis experiment." For inducible expression, cells
were grown in Hartley medium," otherwise LB
(Luria Broth) was used. The construct barnasebarstar has been previously cloned in plasmid
pMT416 by Hartley and Paddon" and this recombinant plasmid was obtained from Dr. Hartley. The
plasmid pMa/c used for the mutagenesis and the expression of barnase was obtained from Dr. P. Stanssens.1°
The expression cassette of barnase consists of the
structural gene, the tuc promoter and phoA signal
sequence. Hence the expression is induced with
IPTG and secreted into the periplasmic space. An
acid shock for separation of the periplasmatic material and the first two chromatografic purification
steps with SP-Trisacryl and a Mono S column, were
performed as described by Mossakowska et al.5 Further purification was achieved on the Pharmacia
FPLC-system with a prepacked Phenyl Superose
column. Barnase was dialyzed overnight against 50
+
+
K. BASTYNS ET AL.
372
TABLE I. Molar Extinction Coefficients (E) Used to Calculate the Concentrations
of the Substrates, and Change in Molar Extinction Coefficients (Ac)Used to
Calculate the Activities
Substrate
PH
GPC
Poly(A)
7
RNA
6.2
7.5
A
280
260
260
E
(M-lcrn-l)
PH*
12.6OOzs
5
10.00029~t
8.000309t
6.2
AE (M-lcrn-l)
A*
280
260
298
8
2.130"
5.0OOz9
-39*
*pH and at which Ae was determined.
+Expressedas formal mononucleotidemolarity.
*Owndetermination at pH 8.0.
mM imidazole-HC1 pH 6.6 containing 1.7 M
(NH,),SO,. The protein was loaded onto the Phenyl
Superose column preequilibrated with the same
buffer. Barnase was eluted with a linear gradient of
50 mM imidazole-HC1 pH 6.6 and then dialyzed
against an appropriate buffer. After further concentration of the purified sample, the homogeneity of
barnase was checked with SDS-polyacrylamideelectrophoresis (Midget system, Pharmacia).
The concentration of the enzyme and substrates
were determined spectrophotometrically. The concentration of the enzyme was determined at 280 nm
with the extinction coefficient rZsonm = 27.411
M-l.cm-' calculated from the amino acid composition (3 Trp and 7 Tyr) and the molar extinction coefficients for Trp and
The molar extinction
coefficient corresponds to a value of 2.18 cm'mg-l.
The concentration of GpC, poly(A) and RNA were
determined using the molar extinction coefficients
given in Table I. It should be noted that the concentration of poly(A) and RNA is thus expressed as the
formal concentration of mononucleotides.
The steady state kinetic studies of the cleavage of
the substrates GpC, poly(A), and RNA were performed with a Kontron Uvicon 81OP spectrophotometer. All the kinetic measurements were performed
at 25°C. The kinetic parameters were calculated using the changes in molar extinction coefficients Ar
given in Table I.
The poly(A) cleavage reaction was followed by an
increase in absorption at a wavelength of 260 nm.
The change in absorption upon complete digestion
(A raBOnm)
was determined.
The kinetics of RNA cleavage by barnase were
studied by following the decrease in absorbance a t
298.5 nm.14 An amount of Torula yeast RNA was
dissolved in distilled water. After filtration (0.22
Km) the solution was extensively dialysed (cut-off =
13,000) to remove short fragments. The RNA stock
solution was then diluted into the appropriate
buffer, just before the kinetic measurements. The
change in absorbance upon complete digestion was
experimentally determined.
Where the pH dependence was determined the following buffer systems were used: formic acid-NaOH
(pH 3.0-4.51, acetic acid-NaOH (pH 4.5-5.8), imid-
azole-HCl (pH 6.0-7.Q and Tris-HC1 (pH 7.8-9).
In these pH-dependency studies, the ionic strength
was always 0.1 M. No appreciable discontinuities
were observed between buffer systems of comparable pH.
The hydrolysis of the cyclic intermediate was followed by the pH-stat method. A radiometer titrator
TTTla and a titrigraph SBR 2C were used to add
NaOH (1 mN) during the hydrolysis reaction. The
substrate was dissolved in a 5 mM imidazole-HC1
buffer a t pH 6.5 and the ionic strength was made 0.3
M with Na,SO,. The substrate concentration was
0.2 to 3.5 mM, and the reaction was started by adding barnase to a concentration of 10 pM. The total
volume of the solution was 700 pL. The solution was
stirred continuously and a micro-electrodewas used
to measure the pH. The stoichiometry was calculated using a pK, of 6.0 for the second phosphate
ionization of 3'GMP.15
Isoelectrofocussing was done with Immobiline Dry
strips pH interval 3.5-10 on a precooled (15°C)Multiphor I1 electrophoresis unit (Pharmacia). A commercial calibration mixture was used.
The conformational stability of the wild type and
the Asp mutants was checked by measuring the ratio of the fluorescence intensities y = 13,0nm/1330nm
after excitation a t 295 nm, as a function of urea
concentration (data not shown). The transition
curves obtained can be described by the following
equation:
(YN
Y=
+ ~ N . [ D ]-t) ("D + ~D.[D]).K
l + K
with
K
=
[Ul/[N] = exp (-
AGO(H20)
- m[D])/RT
where yN (yD)is the relative fluorescence of the native (denatured) form, mN (m,) is the intrinsic
change of this parameter with denaturant (D) in the
native (denatured U) state, m is the cooperativity of
the transition, K is the equilibrium constant for denaturation and AGD(H20) is the free energy of denaturation in water. Wild type Barnase was characterized by K = 1a t [urea],, = 4.64 M and m = 2374 +.
70 kcaUmo1.M. All Asp-mutants had a [urea],, =
373
ELECTROSTATIC EFFECTS IN BARNASE
TABLE 11. Isoelectric Point (PI)for Wild Type
and Mutant Barnase as Determined by
Isoelectric Focusing (pIeF,,) or by
Calculation (pIcd) Accordmg to the
Tanford-Kirkwood Model*
Protein
Wild-type
S85E
S85E,D86N
S85D
S85D,D86N
E60Q
PI,,,
9.20 ? 0.22
7.97 f 0.11
9.30 f 0.22
7.76 2 0.11
8.70 f 0.22
9.45 f 0.16
PIC,,
9.3
7.9
9.25
8.10
9.25
9.6
*Ionic strength was 0.01 M.
*
4.65
0.05 and a cooperativity m = 2314 2 380
showing that the stability of the mutants was not
significantly different from the wild type protein.
All curve fittings were done using SigmaplotR
(Jandel Scientific, Erkrath, Germany).
Electrostatic Calculations
The DELPHI-program was used for the macroscopic electrostatic calculations.16In the interior of
the protein a dielectric constant of 4 was used,17
while the external value was 78.5. To the X-ray coordinates of barnase H-atoms were added with the
aid of the Brugel-programme. In the initial model of
the mutants the same x1,x2, and x3 angles are used
as in the wild type. Then the added atoms were energy minimized using the CHARMM force field. The
allowed range of Apk, is calculated using the extremes of the charg-harge distances of the different allowed rotamers of xZ and x3 for Glu and of x2
for Asp. In the case of the Asp mutants additional
molecular dynamics calculations were performed
(see further).
For the calculation of the isoelectric point, the solvent accessible Tanford-Kirkwood model was applied." The charged groups present in barnase are
tabulated by Loewenthal et a1.l'
Molecular dynamics calculations were performed
using the GROMOS 87 package:'
which was obtained from Biostructure S.A. (Strasbourg, France).
The input files for the GROMOS 87 package were
mainly generated using the program WHATIFFl
which was also used for the visualization of the calculated results and the generation of the structure of
the mutants.
The energy of the structure was first minimized
for 100 steps in vacuum (steepest descent"), and
then placed in a truncated octahedral box of SPC
wateP3 where a minimum distance of 8 A was kept
between the protein and the border of the box. This
results in a 61.78 A wide water box with 3,403 water
molecules. We counted as charged residues Arg+ ,
Lys+,His+, Glu- and Asp-. Counterions (C1-) were
added to compensate for these charges. The energy
of the molecule was again minimized for another
1000
I
I
I
I
I
I
I
I
I
3
4
5
6
7
8
I
I
800
'
h
'2-
600
v
z
Y
\
;
400
1
u
200
01
DH
Fig. 2. pH profile of the parameter k&K, for the S85E mutant
with the substrate GpC. Ionic strength was 0.1 M. Temperature
was 25°C. The dotted line represents the least squares fit of the
data to equation (1). The parameters obtained are given in Table
111.
500 steps in the water box. The velocities of the atoms were assigned following a maxwellian velocity
distribution a t 100 K. The system was warmed up in
five consecutive steps of 1 ps to 300 K while the
position of the protein atoms were harmonically restrained to their startup positions. Then a free molecular dynamics simulation was performed for 50
ps using a constant pressure of 1bar and a constant
temperature of 300 K. The calculations were performed using an Indy Silicon Graphics workstation.
The data were analyzed using the programs
WHATIFZ1and SIMLYS.24
RESULTS AND DISCUSSION
Isoelectric Point
The results of the experimental determination of
the isoelectric pH for wild-type barnase and the mutants using the IEF-technique are shown in Table 11.
The values calculated using the Tanford-Kirkwood
model are also given.
Introducing an extra charge by changing a Ser
into an Glu at position 85 causes the PI of the protein
to decrease by about 1.2 pH units. Moving a charge
on the surface by the simultaneous removal of
Asp-86 and the introduction of Glu-85, causes no
change of the PI. An almost perfect agreement can
be found with the calculated PI values.
When Asp is introduced as the additional charge
a t position 85 instead of Glu, the experimental PI
decrease is, however, more pronounced. When additionally Asp-86 is changed into Asn-86, a decrease of
the PI with 0.5 pH units is observed. Clearly this
cannot be accounted for by the calculations.
The isoelectric point of our previously studied
374
K. BASTYNS ET AL.
TABLE 111. Comparison of the ApK,, Determined From the p H Profile of k,,dK,
for
the Hydrolysis of GpC, and the ApK,, Calculated According to Gilson and Honig.ls*
Protein
Wild-type
S85E
S85E,D86N
S85D
S85D,D86N
PKa,
4.21 k 0.26
3.90 k 0.25
4.14 0.36
3.92 0.32
3.97 k 0.34
*
*
PKa,
5.56 ? 0.07
6.36 0.11
5.97 k 0.15
6.53 k 0.10
6.87 f 0.04
APK,, (exp)
*
ApK,, (call
-
-
0.80 k 0.18
0.41 0.22
0.97 ? 0.17
1.31 0.11
1.0-0.6
0.6-0.11
1.04-0.86
0.54-0.36
*
*
*The ionic strength was 0.1 M. The error on ApKacexp,is determined by summing the error of the individual pK,
values, while the range of ApK,,,,,, is obtained by taking into account all the sterically allowed rotamers of xz
and x3 for Glu and xz for Asp.
/
E
PHE 82
A
B
Fig. 3. Stereo view of the structure of the wild-type protein (A) and the double mutant ( 8 )after
50 ps of equilibration. The position of His-102, Phe-82, are largely different in the double mutant,
and the hydrogen bridge between His-I02 and Gln-104 is clearly visible.
E60Q mutant was also determined and calculated.6
Here the isoelectric point is increased to 9.60 by the
removal of a charge. Also here the agreement between calculations and experiment is very good.
pKa-Values of Wild Type Barnase
and Mutants
for the hydrolysis of GpC, and were also calculated
using the model of Honig. The assignments of the
observed pKa to Glu-73 and to His-102 was done
before5 on the basis of site directed mutagenesis and
NMR measurements.
The pH profile of the parameter kc,&,
for the
hvdrolvsis is bell-shaDed. Since the substrate does
not show any ionization in the pH region studied:
the bell-shaped curve reflects the titration of active
"
Shifts in the pKa of His-102 were experimentally
determined from the pH dependence of the kc,&,
I
375
ELECTROSTATIC EFFECTS IN BARNASE
>
0.16
0.08
1
700
0.00
0
100
200
300
400
500
600
[ G P C I (uM)
Fig. 4. Michaels-Menten curve for wild-type barnase (0). and
the mutants S85E,A86N (o), and S85E (0)for the substrate GpC.
The pH was 6.5, the ionic strength was 0.1 M, and the temperature was 25°C. The parameters obtained by nonlinear least
squares fitting to the Michaels-Menten equation are shown in
Table IV.
groups on barnase. The bell-shaped curve can be described with an equation that takes into account the
ionization of two groups:
vmax
KM
(2)
(
0
1
1+-+-)
[H+l
K1
Kz
(1)
[H+l
These are presumed to be Glu-73 (with ionization
constant K,) and His-102, with ionization constant
K,.5 (VmJKM)o is the pH independent maximum.
The parameters can be obtained by fitting the data
to equation (1) using the general non-linear least
squares curve fitting program Sigmaplot. A typical
curve is shown in Figure 2. These experiments were
repeated three times and the average values and the
errors of the acid dissociation constants are shown in
Table 111. The error of pk,, of Glu-73 is much larger
than of the pK,, because a smaller number of ex-
perimental points was determined in the acid pH
range.
The ApK,, values were calculated using the DELPHI algorithm at an ionic strength of 0.1 M and a
uniform dielectric constant of the protein of 4 was
used.17
The agreement between the calculated and experimentally determined values for ApK,, is very good
for the Glu mutants. When an extra charge (Glu) is
built in the protein, the pK,, rises with 0.8 (t 0.2)
units, when the charge of residue 86 is moved closer
to His-102, the pK,, rises with only 0.4 ( 2 0.2) units.
When Asp is introduced at position 85, the experimentally observed increase of ApK,, is still in
agreement with the calculated one, but in the case of
the double mutant, the experimental increase is
about 0.7 units larger.
The deviating result for the S85D, D85N double
mutant indicates some complications which are not
seen by the structure calculation procedures used.
The overall stability of the mutant, however, is the
same as the wild-type as deduced from the similar
value of the [urea],, concentration and the cooperativity parameter. Therefore, a molecular dyngmics
calculation was applied to the Asp-mutants and-the
wild type as reference.
Although the molecular dynamics calculations
were only performed for 50 ps, the system evolves
very rapidly towards a new conformation in which a
hydrogen bridge is formed between Asp-85 and Asn86. This reorganization seems to have a strong influence on the backbone bearing His-102. This residue rotates out of his normal position to make a
hydrogen bridge with Gln-104, and Phe-82 rotates
towards the hole previously occupied by His-102
(See Figure 3). The additional hydrogen bridge stabilizes the protonated form of His-102 and could be
responsible for the unexpected rise of the pK,. An
extra rise of the pK, of 0.7 units corresponds to a
AGO = 1 kcal/mole, which is very reasonable for a
hydrogen bridge.25 Although this conformation is
not necessarily the only conformation accessible, a
contribution from it explains the observed deviations in the electrostatic calculations. The swinging
out of His-102 is most certainly a dynamic process
TABLE IV.Kinetic Parameters for the Transesterification of GpC (I = 0.1 M) and
the Hydrolysis of the Cyclic Intermediate G > p (I = 0.3 M)*
Protein
Wild-type
Substrate
GPC
kCatW1)
0.38 2 0.03
Wild-type
GPC
GPC
GP'
S85E
S85E,D86N
G>P
G>P
0.15 f 0.03
0.19 f 0.03
0.22 f 0.04
0.26 f 0.05
0.14 f 0.01
S85E
S85E,D86N
&(dW
652 2 91
858 -t 150
689 -t 130
1840 f 550
2390 f 800
2280 f 400
kcat/KM(M-ls-')
582 f 127
175 2 65
276 f 95
119 -t 57
109 f 57
61 k 15
I
*Temperature = 25"C,pH = 6.5. The errors for K,, and K, were calculated using the nonlinear fitting
program Sigmaplot, and the errors for kJK, were calculated using the previous errors by applying the
rules for the propagation of errors.
376
K. BASTYNS ET AL.
which is reversed in the unprotonated state since
the enzyme is active with dinucleotides.
I
I
I
I
I
A Comparison of the Kinetic Parameters for
Dinucleotides and Cyclic Intermediates
The kinetic parameters can be obtained from the
full Michaelis-Menten curve which is shown in Figure 4 for dinucleotide substrates. These values were
obtained at 6.5 pH. The parameters obtained by fitting the data to the Michaelis-Menten equation are
shown in Table IV. Only the Glu mutants are studied in detail. The parameters change but not dramatically. The change is most pronounced in
kCat/KMof the S85E mutant which decreases by a
factor of 6 for GpC In all other situations the
changes are smaller.
The kinetics of the hydrolysis of the cyclic intermediate were studied using the pH-stat method. The
characteristic parameters are found in Table IV.
Here again the changes of the catalytic parameters
are relatively limited.
Hydrolysis of Polymeric Substrates
The activity of barnase WT and mutants is also
determined with the natural substrate RNA and the
artificial substrate poly(A). Barnase is a much better enzyme for RNA due to the presence of subsitesz6 The pH optimum for RNA is a t pH 8.0 instead of pH 5 for the dinucleotides. From Figure 5
and Table V it can be deduced that WT barnase and
the double mutant have kCat/K, values that are
comparable. However, the ,
k and KM values of the
double mutant are two times larger than the corresponding parameters of the wild type. In contrast,
the S85E mutant has a KMthat is dramatically increased. In fact, for this mutant only the parameter
kcat/KMcan be calculated. This value is decreased by
a factor of 13.
For poly(A) (measured at pH = 6.2) a similar
overall situation is found, except that for the single
mutant all the parameters could be determined.
Here it is clear that KM increases by a factor of 9.2.
The question can be asked whether the strong increase of the KM for polymeric substrates due to the
Fig. 5. Michaelis-Menten curve for the hydrolysis of RNA by
the wild-type enzyme (a), and the mutants S85E,A86N (o),and
the mutant S85E (0).
pH = 8.0, I = 0.1 M, temperature = 25°C.
The parameters obtained by nonlinear least squares fitting to the
Michaelis-Menten equation, or to a straight line, are shown in
Table V.
introduction of the extra negative charge a t position
85 can be explained on the basis of the overall
charge of the protein. The experiments with RNA
seem to indicate this. These experiments were done
at pH 8.0 where WT barnase is positively charged
(PI = 9.3) while the S85E mutant, which has an
experimental PI of 7.97 shows a charge that is almost zero. However, the same deleterious effect of
the extra charge is also observed for poly(A) at pH
6.2, despite the fact that both proteins have a net
positive charge.
It is therefore clear that the observed deleterious
effect of this extra charge on the KM of polymeric
substrates is due to a local effect only visible with
polymeric substrates. In fact, when the structure of
the mutants, as obtained from molecular dynamics
calculations, is compared to the structure of the barnase-tetranucleotide complexz7 it becomes clear
TABLE V. Kinetic Parameters for the Wild-Type and Mutant Barnase for the Natural
Substrate RNA (pH = 8 ) and the Artificial Substrate Poly(A) (pH = 6.2)*
Substrate
kcat (S-')
KM (PM)
kCat&q(PM-~s-')
RNA
3998 f 870
2130 f 560
1.87 f 0.9
RNA
0.15 f 0.01
RNA
6765 f 790
3412 f 1100
1.98 f 0.9
poly(A)
26.7 f 1.2
263 f 30
0.12 f. 0.02
2415 f 340
0.007 f. 0.002
poly(A)
17.6 f 4.0
406 f 42
0.094 f 0.014
poly(A)
38.6 f 1.7
*Temperature = 25"C,I = 0.1 M.The errors fork,,, and K, were calculated by Sigmaplot, and for k J K , were
calculated by the rules for the propagation of errors. In the case of the mutant S85E and RNA as substrate, only
Protein
Wild-type
S85E
S85E,D86N
Wild-type
S85E
S85E,D86N
k,,JK, could be determined because a linear relation between v and the substrate concentration was obtained.
For this substrate the errors for k,,dKM were obtained from linear least squares fitting to the 16 data points.
(Mean values and standard deviations are given.)
377
ELECTROSTATIC EFFECTS IN BARNASE
that the extra negative charge points towards the P2
site. This explains immediately its specific effect on
the polymers.
CONCLUSION
Mutations were made with the goal to introduce
an extra charge or to move a charge on the surface
closer to His-102. The effect of these mutations on
the isoelectric point of the enzyme can be calculated
with the Tanford-Kirkwood theory and when Glu is
used to replace Ser-85 the correlation with the experimental values is perfect. However, when Asp is
used, the correlation is less.
The effect on the pK, of His-102 can be determined from the pH dependence of the k,,,/KM for the
hydrolysis of dinucleotides, e.g., GpC. The effect can
also be calculated using the the method of Honig.
Again the agreement is very good for the Glu mutants and the single Asp-mutant, but less for the
double Asp mutants.
The aberrant behavior of the double Asp mutant
becomes clear from molecular dynamics calculations: the protonated state of His-102 is stabilized by
a new hydrogen bridge with the oxygen of Gln-104.
This hydrogen bridge becomes possible thanks to a
reorientation of the His-102, itself due to another
new hydrogen bridge between Asn-85 and Asp-86.
The reorientation of the His ring also explains the
lower activity of the enzyme.
The effect of the Glu-mutations was also tested on
all the kinetic parameters for GpC, G > p, at pH 6.5,
for RNA at pH 8.0, and for poly(A) a t pH 6.2. The
effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong
increase of the KM is observed in the case of the
single mutant (extra negative charge) and the polymeric substrates.
These results prove that the KM of barnase for
polymeric substrates is strongly influenced by electrostatic charge effects and these effects are equally
operative in the ground as in the transition state.
We suggest that this deleterious repulsion is operating on P2, in view of the position of Asp-86 relative to the structure of the barnase-tetranucleotide
complex proposed by Buckle and Fersht.”
ACKNOWLEDGMENTS
This research was supported by the Belgian national incentive program on fundamental research
in life sciences initiated by the Belgian State, Prime
Minister’s Office, Science Policy Program (Bioimpuls/O5) and by the Research Council of the University of Leuven. G.V. is supported by contract
ETC-007 of the “Vlaams Actieprogramma Biotechnologie.” The authors thank Dr. R.W. Hartley
(N.I.H., Bethesda, MD) and Dr. P. Stanssens (Plant
Genetic Systems, Gent, Belgium) for the gift of the
expression system, and B. Keyers, C.E. for starting
up the expression system. The authors thank Dr. Y.
Mauguen for the availability of the coordinates of
barnase and Dr. S. Wodak (University of Brussels,
Belgium) for the use of the DELPHI program. Prof.
J. Vanderleyden (F.A. Janssens Laboratory for Genetics) is acknowledged for making available the
isoelectrofocusing unit. Dr. B. Wroblowski is acknowledged for valuable discussions.
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