ABSTRACT
Based on crystal structure analysis of the
Serratia
nuclease and a sequence alignment of six related nucleases, conserved amino acid
residues that are located in proximity to the previously identified catalytic
site residue His89 were selected for a mutagenesis study. Five out of 12 amino
acid residues analyzed turned out to be of particular importance for the
catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their
replacement by alanine, for example, resulted in mutant proteins of very low
activity, <1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that
some of these mutants are predominantly affected in their
k
cat, others in their
K
m. These results and the determination of the pH and metal ion dependence of
selected mutant proteins were used for a tentative assignment for the function
of these amino acid residues in the mechanism of phosphodiester bond cleavage
by the
Serratia
nuclease.
Serratia
nuclease (EC 3.1.30.2) catalyzes the hydrolytic cleavage of DNA and RNA between
the 5'-phosphate and the 3'-oxygen of the sugar moiety in the presence of Mg
2+
or several other divalent metal ions (
1
,
2
). The enzyme is able to cleave both single- and double-stranded substrates with similar efficiency, but like other non-specific nucleases shows certain sequence preferences (
3
,
4
). The gene of the nuclease has been cloned (
5
) and protein sequence alignments (
6
,
7
) have shown that this enzyme belongs to a new class of non-specific nucleases from different species, namely
Saccharomyces cerevisiae
(
8
),
Anabaena
sp. 7120 (
9
),
Syncephalastrum racemosum
(
10
),
Bos taurus
(
11
) and probably also
Streptococcus pneumoniae
(
12
). Although significant sequence homology exists among these proteins, the
biological functions of these nucleases are quite distinct, ranging from an
apparently simple nutritional function for the extracellular
Serratia
and
Anabaena
nucleases, involvement in DNA repair or recombination in the case of the endo-exonuclease from yeast (
6
), to generation of primers for mitochondrial DNA replication (
11
). For this group of proteins the
Serratia
nuclease has become the paradigm, as many biochemical and biophysical studies
have been performed on this enzyme, in particular the recently published 2.1 Å X-ray crystal structure analysis (
13
,
14
). Although the structure was solved for the free enzyme, i.e. in the absence of
a metal ion cofactor and substrate, several lines of evidence led to the
proposal that the active site is located around His89 and Glu127, residues
which are conserved among the homologous nucleases (
13
). The independently carried out mutational analysis of this enzyme in which
several conserved amino acid residues were substituted by Ala confirmed this
proposition (
7
). The two mutants with the lowest residual activity were H89A and E127A,
showing <0.001 and 0.1% respectively of the activity of the wild-type enzyme. However, based on these data it was neither possible to
propose a mechanism of nucleic acid cleavage nor to assign a role for these two
residues in catalysis. Moreover, as the unique tertiary fold of the
Serratia
nuclease and the structure of the presumptive active site suggests that the
mechanism of nucleic acid cleavage is different from other nucleases of known
structure, arguments by analogy for a likely mechanism of action could not be
presented.
In this paper we report the results of biochemical experiments designed to
identify the catalytic function of several residues within the presumptive
active site of the
Serratia
nuclease. To this end we have extended our previously published mutational
analysis to all residues conserved among the six homologous nucleases and
located those that can be considered candidates for involvment in catalysis.
The mutational analysis presented here was guided both by the X-ray structure and by an improved and expanded sequence alignment. It
includes an investigation of the pH dependence and of the metal ion cofactor
requirement of selected mutants. Based on the results presented here we put
forward a suggestion for a likely mechanism of action of this enzyme.
Chemicals for electrophoresis were purchased from Gibco BRL or Baker, chemicals
for oligonucleotide synthesis from Millipore. All other chemicals were supplied
by Merck. Restriction enzymes were obtained from Amersham-Buchler or Boehringer-Mannheim. T4 polynucleotide kinase, T4 DNA ligase and
Taq
DNA polymerase were from Amersham-Buchler,
Pfu
DNA polymerase from Stratagene. All enzymes were used according to the
respective manufacturer's recommendations. Salmon testis DNA and herring sperm
DNA were from Sigma and Pharmacia respectively.
Oligodeoxyribonucleotides were synthesized on a Millipore Cyclone Plus DNA
synthesizer using standard [beta]-cyanoethylphosphoramidite chemistry (
15
) on the 0.2 [mu]mol scale. Synthesis was carried out in the trityl-off mode and deprotection was performed using established protocols.
Purification was performed by standard denaturing gel electrophoresis and the
purified oligodeoxyribonucleotides were desalted by gel filtration using NAP-10 columns.
Protein sequences of
Serratia
nuclease (NUC_SERMA from the SWISS-PROT Protein Sequence Database),
Saccharomyces cerevisiae
mitochondrial endo-exonuclease (NUC1_YEAST),
Anabaena
sp. nuclease (NUCA_ANASP),
Bos taurus
endonuclease G (NUCG_BOVIN), DNA entry nuclease from
Strep- tococcus pneumoniae
(NUCE_STRPN) and a partial sequence of the deoxyribonuclease
from
Syncephalastrum racemosum
(PIR S33276 from PIR Protein;
10
) were aligned using CLUSTAL W 1.5 (
16
) with the standard parameters (for details see
7
). The alignment was refined after comparison with the X-ray structure of the
Serratia
nuclease (
13
).
Site-directed mutagenesis was performed using a two step PCR mutagenesis
protocol essentially as described in detail before (
7
). After PCR, the mutated gene was cloned into the
Bam
HI and
Sal
I-cleaved plasmid pHisNuc and transformed into
Escherichia coli
LK111([lambda]) (
17
).
Mutant clones were identified after PCR amplification of the
Serratia
gene starting with DNA from single colonies, followed by digestion of the PCR
product by restriction enzymes specific for silent marker sites introduced
together with the codon exchange. After plasmid preparation (QIAGEN Plasmid
Mini Kit), the mutation was verified by DNA sequencing of the whole
Serratia
nuclease gene.
Plasmids containing the gene for the His
6
-tagged
Serratia
nuclease (wild-type or mutant) were transformed into
E.coli
TGE900 (
18
). Cells were grown at 28oC, heat induced at 42oC and the nuclease extracted from inclusion bodies by urea treatment
and purified to homogeneity by Ni-NTA affinity chromatography essentially as described before (
19
). Protein concentrations were determined using the extinction coefficient for
the isoenzyme SM2 of [epsilon]
280
= 47 292 M
-1
cm
-1
(
20
). For Y76F a theoretical extinction coefficient of [epsilon]
280
= 43 100 M
-1
cm
-1
, based on the content of Trp, Tyr and Phe, was used (
21
). The enzyme with the same amino acid sequence as isoenzyme SM2 (
22
) except for the N-terminal Met-(His)
6
-Gly-Ser affinity tag is referred to as wild-type throughout the following text.
CD spectra of the
Serratia
nuclease at a concentration of 5 [mu]M were recorded in a Jasco J-710 model dichrograph from 185 to 250 nm at 16oC in a cylindrical cuvette of 0.05 cm path length in 10 mM Tris-HCl, pH 8.2. After recording the CD spectrum of the
protein, the baseline given by the CD spectrum of 10 mM Tris-HCl, pH 8.2, was recorded and subtracted from the protein CD spectrum.
The activity of wild-type and mutant
Serratia
nuclease preparations was determined using high molecular weight DNA from
salmon testis (Sigma D-1626) or herring sperm DNA (Pharmacia 27-4564-01) as substrate. Experiments were carried out in 10 or 1 mm
(for DNA concentrations >0.1 mg/ml) cuvettes at 25oC with a Hitachi U-3000 spectrophotometer set at 260 nm. Substrate DNA concentrations
were varied between 0.005 and 1 mg/ml (~15 [mu]M to 3 mM nucleotides). Concentration of substrates (in nucleotides)
were calculated using e
260
of 6600 M
-1
cm
-1
for double-stranded DNA. As for DNase I, the specific activity of the
Serratia
nuclease can be expressed in terms of Kunitz units (KU) per mg protein (
23
). 1 KU is defined as the amount of enzyme needed for an increase of 0.001 A
260 nm
/min at 25oC in a 1 ml volume at 1 cm path length. For determination of the apparent
rates of reaction, full progress curves were recorded and the maximum rate
expressed as [Delta]A
260
/min was determined. Measurements were performed in a volume of 120 [mu]l. After pre-warming the solution containing all components but the nuclease for 1 min, the reaction was started by addition of 1-10 [mu]l appropriately diluted wild-type or mutant enzyme to obtain maximum velocities of
<= 10%/min of the total hyperchromic effect.
For the Mg
2+
ion dependence of DNA cleavage by wild-type and mutant proteins, the substrate DNA was dialyzed extensively
against 10 mM Tris, 1 mM EDTA, pH 8.0 to remove all traces of divalent metal
ions and thereafter against 10 mM Tris-HCl, pH 8.0. Aliquots of 180 [mu]l nuclease at appropriate concentration (for example wild-type 0.5 nM, D86A 70 nM and E127A 600 nM) and DNA (0.075 mg/ml)
were pre-incubated at 25oC in 40 mM Tris-HCl, pH 8.0, and NaCl at concentrations to give a constant
ionic strength of 0.1 M. The reaction was started by addition of 20 [mu]l MgCl
2
solution of appropriate concentration.
The change in absorbance accompanying DNA hydrolysis was used to convert
velocities measured as [Delta]A
260
/min to
k
cat
units (s
-1
). The hyperchromic effect occurs when double-stranded DNA is separated into individual strands and/or cut to small
single-stranded oligonucleotides <= 10 nt in length (
24
). For example, the increase in absorbance at 260 nm for salmon testis DNA after
complete hydrolysis with
Serratia
nuclease is 33%. This increase in absorbance is proportional to the decrease in
chain length for a substantial part of the reaction. The change in
hyperchromicity can, therefore, be converted to the rate of phosphodiester bond
cleavage, similarly to as in the report of Hale
et al
. (
25
):
For evaluation of the pH dependence of the
Serratia
nuclease-catalyzed cleavage of nucleic acids we used the following buffer
substances (p
K
a
, pH range): NaAc (4.75, 3.5-5.8); HEPES-NaOH (7.55, 6.5-8.6); Tris-HCl (8.30, 7.2-9.6); CAPS-NaOH (10.4, 9.0-10.9). All buffers were made
from stocks containing 180 mM NaCl, 10 mM MgCl
2
, 20 mM buffer substance by mixing the base and the acid form of the buffer
substance, e.g. sodium acetate with acetic acid. pH was measured at 20oC using a pH meter 761 (Knick) equipped with a combination electrode type
N6280 (Schott) calibrated with standard buffer pH set 1 (Merck). Variation in
ionic strength was <10% and controlled by measuring the conductivity with a Schott conductometer CG
855 equipped with an LF1100 electrode. Cleavage kinetics were recorded using a
microtiter dish assay by measuring the disappearance of ethidium stained DNA
fluorescence upon cleavage of DNA. The method will be described in more detail
elsewhere. Briefly, 0.025 mg/ml salmon testis
DNA in 10 mM buffer, 90 mM NaCl, 5 mM MgCl
2
and 5 [mu]M ethidium bromide were incubated in a 100 [mu]l volume at room temperature with the nuclease. The ethidium stained DNA
fluorescence was monitored after excitation at 312 nm on a transilluminator
with a video documentation system (INTAS, Göttingen, Germany).
The active site of the
Serratia
nuclease has been located by crystallographic analysis of the enzyme (
14
) and by an alignment-guided mutagenesis approach (
7
). According to these studies His89 is part of the active site and essential for
catalysis. It is surrounded by several amino acid residues conserved among a
group of non-specific nucleases (Figs
1
and
2
). Mutagenesis of some of these residues to Ala had shown that Asp86, Arg87 and
Glu127 are also strong candidates for being involved in catalysis; their
specific functions, however, have not yet been analyzed. Based on a new
multiple sequence alignment containing six nucleases and, more importantly, by
using the detailed information of the 3-dimensional structure of the
Serratia
nuclease (
14
) we have extended our previous mutational analysis (
7
) to characterize amino acid residues likely to be involved in catalysis and to
analyze their role. The new alignment shows that a substantial number of
residues are conserved in all five nucleases (for the sixth nuclease only
partial sequence information is available). We therefore included in our
mutational analysis all conserved functional amino acid residues located in the
direct spatial neighborhood of His89 (Figs
1
and
2
and Table
1
). All amino acid residues but Tyr76, which was substituted by Phe, were changed
to Ala and several of them also for other, usually similar, amino acid
residues. Of 27 mutants analyzed here, 21 had to be produced and six were
already available from a previous study (
7
). Mutant proteins were purified to homogeneity and analyzed for the integrity
of their (secondary) structure by CD spectroscopy. The nucleolytic activity of
the mutants was determined and, where possible, steady-state parameters were evaluated for the cleavage reaction. Mutants with
substitution of amino acid residues considered to be involved in acid-base catalysis and/or Mg
2+
binding were also analyzed with respect to their pH dependence and metal ion
requirement.
The homology between the
S.marcescens
,
S.cerevisiae
,
Anabaena
sp. and
B.taurus
nucleases has been reported before (
6
,
7
,
9
). The nuclease of
S.racemosum
was considered to be homologous to DNase I, although it has only 12 of 40 N-terminal amino acid residues in common with DNase I (
10
). However, as can be seen from Figure
2
, this nuclease shares 16 identical residues with the nuclease of
S.cerevisiae
and, more importantly, an internal tryptic fragment comprising nine amino acids
has seven amino acids in common with this nuclease but no homology to DNase I.
We therefore believe that this nuclease fits well into the class of DNA/RNA non-specific endonucleases. The extracellular nuclease of
S.pneumoniae
was included in this alignment, although the overall similarity to the other
nucleases is low, because most of the conserved amino acid residues located
around the active site of the
Serratia
nuclease are conserved in this nuclease, including all residues of the PROSITE
motif D-R-G-H-[QIL]- X
3
-A (accession no. PDOC00821), characteristic of the family of DNA/RNA non-specific endonucleases. Incident- ally, no other proteins homologous to the
S.pneumoniae
nuclease were found in the SWISS-PROT databank.
We have produced 27 mutants of the
Serratia
nuclease at 12 different positions by site-directed mutagenesis and overexpression in
E.coli
(see Table
1
for an overview). All mutant proteins were purified to homogeneity. They were
obtained as soluble proteins, although some of them, upon incubation with DNA,
showed a tendency to aggregate. As judged from their CD spectra all mutants are
likely to have a similar structure to the wild-type enzyme. The CD spectrum of K172A was the only one to show a minor
deviation from the CD spectrum of the wild-type nuclease (data not shown), which could be due to the fact that this
residue is part of the central [beta]-sheet.
The cleavage activity of the mutants was measured with the hyperchromicity assay
using salmon testis DNA as substrate (Table
1
). The activity of the mutants H89D and H89Q could not be determined accurately,
as they aggregate rapidly in the presence of DNA.
Mutations at two positions, His89 and Asn119, lead to almost inactive enzymes,
suggesting that these amino acid residues have a very important function in
catalysis. At position 89 only H89N shows measurable activity (0.13%). It is
mainly affected in its
k
cat
. All other mutants (His -> Ala, Asp, Gln, Glu or Lys) with amino acid substitutions introduced at
position 89 show drastically reduced activity (<0.001%). At position 119 all amino acid substitutions tested (Asn -> Ala, Asp, Gln or His) result in mutant proteins with very little nuclease
activity (0.001-0.1%), N119D and N119Q being the most active ones (0.05 and 0.1%
respectively). N119D and N119Q, like H89N, are also much more affected in their
k
cat
than
K
m
.
The next group of mutants with markedly reduced activity contains the amino acid
residues Arg57, Arg87 and Glu127. The large decrease in activity observed upon
substitution by Ala (0.1-0.6%) indicates that these amino acids could be directly involved in
catalysis. Arg57 and Arg87 are thought to be near the phosphate backbone of the
nucleic acid substrate and alterations in these positions could change the
binding and orientation of the substrate (
13
). A similar argument would hold for Glu127, which, via Mg
2+
, could also serve to bind and position the substrate. In these mutants the
substitution of Arg by Ala is more deleterious than by Lys and the substitution
of Glu by Ala or Gln is more deleterious than by Asp. Thus, the conservation of
charge helps to retain some activity, but only in the case of R87K is the wild-type activity almost reached. This result is interpreted to mean that
Arg57 and Glu127 are, in contrast to Arg87, needed for very precise
interactions and therefore most likely are directly involved in catalysis.
Mutants of Arg57 are more affected in their
k
cat
than
K
m
, while the opposite is true for the R87A mutant. With Glu127 amino acid
substitutions lead to mutant enzymes impaired more or less in
k
cat
and
K
m
, depending on the substitution.
Less dramatic effects are observed when the conserved amino acid residues Asp86,
Asn110 and Gln114 are substituted by Ala (~1%). For Asn110 and Gln114 this reduction can be explained by indirect
effects on the catalytic center. These residues, as shown by the X-ray structure, have important structural functions, which include making
hydrogen bonds to either the main chain NH and O at position Ala91 (by Asn110)
of the active site loop containing the D-R-G-H-motif or to the side chain of Asn119 (by Gln114)
respectively. The effects of substitutions for Asp86 (Asp -> Ala or Glu) cannot be rationalized in a straightforward manner by
structural effects. While D86A is a
K
m
mutant, D86E exhibits a decrease in both
k
cat
and
K
m
.
Table 1
Although E211A shows reduced activity (
7
), the X-ray-structure and the results with the two mutants E211D and E211Q, both
having wild-type activity, show that Glu211 is not likely to be directly involved in
catalysis. The structural analysis (
13
) suggests that it has an important structural function by forming a salt link
with Lys231. This electrostatic interaction also includes the participation of
Lys212 via an intervening water molecule. Glu211 is also hydrogen bonded to the
main chain at position Ile218 of [beta]-sheet 2'. This contact presumably can also be made by Asp or Gln, but
not by Ala.
Mutation of the conserved Tyr76 to Phe results in a mutant protein with only
slightly reduced activity. Therefore, the hydroxyl group of Tyr76 is unlikely
to participate in the catalytic action of the
Serratia
nuclease. In particular, it cannot be involved in a covalent intermediate as
observed, for example, with topoisomerases (reviewed in
29
).
Activity versus pH profiles were determined for H89N, N119D, E127Q and E127D.
For the activity of the wild-type enzyme a bell-shaped curve was obtained with an optimum around pH 8. The shapes of
the activity versus pH profiles of E127Q and E127D are similar to that of the
wild-type enzyme (data not shown), while those of H89N and of N119D are
significantly different (Fig.
3
A). In contrast to the pH dependence of the activity of the wild-type enzyme, the activity of the H89N mutant rises almost steadily over
the entire pH range. Comparison of the two profiles suggests that His89 is
engaged in a protonation/deprotonation equilibrium around pH 5.5-6.5 and that protonation interferes with activity of the enzyme. For the
N119D mutant, the profile has a maximum at ~pH 5 which can be rationalized by assuming that deprotonation of Asp119
introduces a negative charge at the active site of the nuclease which is deleterious for nuclease
activity. At low pH Asp119 is protonated and hence could function in a similar
way to Asn119. Indeed, the activity of the N119D mutant approaches the wild-type activity at very low pH.
We investigated the divalent metal ion preference of the nuclease activity of
the wild-type enzyme and selected mutant proteins (D86A, H89N, N119D, E127A, E127D
and E127Q). Only H89N shows a similar divalent metal ion preference to the wild-type nuclease, which is half as active with Mn
2+
as with Mg
2+
. The other mutant proteins are at least twice as active with Mn
2+
than with Mg
2+
, E127A being up to five times more active. Furthermore, the activity of N119D
is dramatically increased with either Co
2+
(20 times) or Zn
2+
(40 times) in comparison to Mg
2+
, while the wild-type enzyme shows equal or reduced activity with Co
2+
and Zn
2+
compared to Mg
2+
. Of particular interest were Asp86 and Glu127, as in proteins Asp and Glu
residues are very often observed as ligands of Mg
2+
. For the mutants D86A and E127A the Mg
2+
concentration dependence is given in Figure
3
B. Both mutants show similar profiles which are offset by a factor of 2-3 towards higher Mg
2+
concentrations compared to the profile of the wild-type nuclease, indicating that these mutants have a slightly lower
affinity for the metal ion cofactor than the wild-type enzyme. A similar result has also been obtained for N119D (data not
shown).
One of the principal goals of an enzymological study is the elucidation of the
mechanism of action for the enzyme under investigation. For a hydrolase like
the
Serratia
nuclease this means in particular identifying those amino acid residues that
are responsible for activation of the attacking water molecule, stabilization
of the transition state and protonation of the leaving group and, based on this
assignment, proposing a reaction mechanism. As for many nucleases, as well as
hydrolases in general, propositions have been published regarding a likely
mechanism of action and reference data and models exist which can be used for a
comparison with the
Serratia
nuclease.
Based on a detailed sequence comparison among related nucleases and a mutational
analysis of conserved amino acid residues and the X-ray structure of the
Serratia
nuclease, we suggest that the active site comprises at least four residues,
namely Arg57, His89, Asn119 and Glu127. These residues are not only located in
the immediate neighborhood of each other (Fig.
1
), but mutagenesis of any of these residues results in proteins with drastically
reduced activity.
The most important residue of the active site seems to be His89. Among the six
H89 mutants only the H89N mutant nuclease shows measurable residual activity.
This mutant is mainly affected in
k
cat
, arguing for a defect in a catalytic function and not in substrate binding.
That His89 is not involved in substrate binding is supported by the finding
that the wild-type protein and H89A bind with similar affinity to a non-cleavable modified dodecamer in which all phosphate residues were
substituted by phosphorothioates (Friedhoff, unpublished results). H89N has a
different pH profile compared to the wild-type enzyme: the p
K
a
of ~5.5-6.5 in the ascending branch of the wild-type activity versus pH profile (Fig.
3
A), which is missing in that of H89N, may therefore be assigned to His89. The
interpretation most consistent with our data is that His89 must be deprotonated
to support catalysis, i.e. to function as the general base. For the H89N mutant
specific base catalysis (OH
-
) would then replace general base catalysis in activation of the water molecule.
A role for His89 as a general acid is unlikely. In this respect
Serratia
nuclease differs from DNase I. For DNase I, which has a pH optimum of 7.5, a
His residue (His134) is considered to be the general acid (
30
). If His89 in the
Serratia
nuclease were the general acid, the question would arise as to which is the
general base. As discussed in Miller
et al
. (
13
), Glu127 could be the general base (rather than His89), however, the relatively
high residual activity of E127Q and the similarities of the pH profiles of the
wild-type enzyme and the E127Q mutant argue against this alternative. RNase T
1
, which uses Glu58 as the general base and His92 as the general acid, has an
optimum around pH 5-6 (
31
).
The substitution of Asn119 by other amino acids is as deleterious for enzyme
activity as substitutions at position His89. Comparison with other hydrolases
of known structure and mechanism offers several possible roles for Asn119. In
RNase A, Gln11 forms a direct contact with the scissile phosphate, which may be
used for transition state stabilization (
32
)
. However, recent mutagenesis results have shown that the RNase A mutants Q11A,
Q11D and Q11H did not have a markedly reduced activity towards natural
substrates (
33
). In DNase I, Asn170 is also seen at hydrogen bond distance from the phosphate
in some structures of this enzyme (
30
) and is also found (Asn153) in the structurally related exonuclease III (
34
). However, no mutagenesis data are available for these residues in the
respective proteins and, as has been shown for RNase A, without such data the
importance of this possible phosphate contact for transition state
stabilization remains speculative. On the other hand, Asn119 in the
Serratia
nuclease might have a similar function to Gln residues (Gln61, Gln200 or Gln204
respectively) in the GTPases p21
ras
(
35
), transducin (
36
) or G
i[alpha]
(
37
) respectively. These residues are involved in positioning the attacking water
and later in stabilization of the transition state. Such a function for Gln or
Asn is also well documented for several proteases, e.g. papain and subtilisin,
were they stabilize the oxyanion in the transition state (
38
-
40
). A plausible interpretation of our data could be that in the
Serratia
nuclease Asn119 is involved in transition state stabilization.
The Ala mutant at position Arg57 is very much impaired in its activity, mainly
due to a large decrease in
k
cat
. This suggests that Arg57, similarly to in other nucleases [Arg77 in RNase T
1
(
41
) and Arg48 in nuclease P1 (
42
)], is likely to be involved in positioning and polarizing the phosphate of the
scissile phosphodiester bond and/or stabilization of the transition state.
The
Serratia
nuclease requires Mg
2+
for phosphodiester bond cleavage. In the presumptive active site only two
acidic amino acid residues are present, Asp86 and Glu127, which are often found
as ligands of Mg
2+
. The cleavage activities of mutants at positions 86 and 127 argue for Glu127 as
the principal ligand of Mg
2+
, as the effects of substitutions by Ala are more severe for Glu127 than for
Asp86. This is supported by the fact that the E127A mutant is stimulated more
by Mn
2+
compared to the D86A mutant. This divalent cation binds more strongly to
multidentate ligands than Mg
2+
and, thus, may compensate for the loss of one ligand as in the Glu127 mutants.
It must be emphasized, however, that N119D and N119Q are also activated by Mn
2+
. It might well be that Mn
2+
can better support cooperative formation of the transition state complex than
Mg
2+
when critical residues needed for transition state stabilization are missing.
Similar observations have been made with the restriction endonucleases
Eco
RI and
Eco
RV (
43
). It is remarkable that N119D, similarly to D86A and E127A, needs a 2-fold higher Mg
2+
concentration for maximum activity than the wild-type enzyme and that its activity is increased 20-fold by Co
2+
and 40-fold by Zn
2+
, while the wild-type enzyme is only stimulated 2- to 3-fold. These results may be considered as an indication that
Asn119 is also involved in metal ion binding.
As we have not been able to identify the general acid needed to protonate the
leaving group, it is tempting to speculate that Glu127 is indirectly involved
in this function by binding the Mg
2+
ion which could associate itself with the leaving group as discussed for the 3' -> 5' exonuclease of the Klenow polymerase (
44
). Alternatively, leaving group protonation could occur at the expense of a Mg
2+
-bound water molecule, as discussed for the restriction endonucleases
Eco
RI and
Eco
RV (
45
).
Three other residues conserved among six related nucleases and located within or
close to the presumptive active site are thought to be involved in substrate
binding, namely Asp86, possibly via Mg
2+
, as indicated by the altered metal ion concentration dependence of the D86A
mutant, Arg87 and Arg131. This conjecture is based on the finding that exchange
to Ala leads to proteins which are mainly affected in catalytic activity by an
increase in
K
m
(between 50- and 100-fold increased
K
m
values compared to between 1.3- and 2.9-fold decreased
k
cat
values).
Starting with the crystal structure of the
Serratia
nuclease and a detailed sequence comparison among the
Serratia
nuclease family of non-specific nucleases, we have carried out a mutational analysis designed to
identify the amino acid residues directly involved in catalysis and to propose
a mechanism of action for this enzyme. Based on the results obtained, the
following mechanism for the hydrolysis of DNA by the
Serratia
nuclease is proposed (Fig.
4
): His89 is likely to function as the general base, which abstracts a proton
from a water molecule which then serves as the attacking nucleophile. By
analogy with many nucleases (
46
), we assume that the attack is in-line with the phosphodiester bond to be cleaved and that the reaction
proceeds without a covalent intermediate. This assumption rests on our finding
that the only reasonable candidate amino acid residue to form a covalent
intermediate, the conserved Tyr76, could be exchanged for Phe without effect on
cleavage activity. The transition state, characterized by a pentacoordinated
phosphorous, could be stabilized by Arg57 and/or Asn119. Glu127 and/or Asn119
could be ligands of the essential cofactor Mg
2+
, which may have two functions, namely to help stabilize the transition state
and to facilitate leaving of the group that carries an extra negative charge
after cleavage of the phosphodiester bond. Leaving group stabilization could
occur by association of Mg
2+
with the terminal phosphate group or by protonation at the expense of a water
molecule from the coordination sphere of the Mg
2+
ion. Several amino acid residues could be indirectly involved in catalysis by
binding to the substrate and positioning the protein backbone for nucleophilic
attack: Arg87 and Arg131 and possibly also Asp86. Asp86 might be engaged in an
attractive interaction via a divalent metal ion or a repulsive interaction,
both types of interaction being in principle useful for a proper orientation of
the phosphodiester backbone. It must be emphasized that the
Serratia
nuclease accepts both DNA and RNA in double- and single-stranded form as substrates; this makes it necessary that these
different substrates are `forced' into a similar conformation by the enzyme to
be acceptable. For this purpose contacts to the sugar-phosphodiester backbone not too far from the phosphodiester bond to be
cleaved are indispensable for the catalytic machinery.
We are grateful to Dr A.Jeltsch and Mr G.Meiss for critical reading of the
manuscript. The expert technical assistance of Ms U.Steindorf is gratefully
acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft
(Pi 122/9-1), the International association for the promotion of cooperation with
scientists from the independent states of the former Soviet Union (INTAS 94-1181) and the Fonds der Chemischen Industrie. KLK acknowledges the support
of the National Institutes of Health, the State of Texas, the Robert A.Welch
Foundation and the Keck Foundation. This paper is part of the PhD requirement
of PF and BK.
Position
Amino acid exchange
k
cat
a
1/
K
m
a
k
cat
/
K
m
a
Wild-type
1 +- 0.09
1 +- 0.25
1 +- 0.33
Arg57
Ala
(1.7 +- 0.1) * 10
-2
(3.7 +- 0.7) * 10
-1
(6.2 +- 2) * 10
-3
Lys
(5 +- 0.3) * 10
-3
12.5 +- 1.4
(6 +- 0.7) * 10
-2
Tyr76
Phe
(3.7 +- 0.1) * 10
-1
4 +- 0.5
1.1 +- 0.2
Asp86
Ala
(7.5 +- 0.5) * 10
-1
(2 +- 0.2) * 10
-2
(1 +- 0.2) * 10
-2
Glu
(1 +- 0.1) * 10
-2
11.1 +- 2.8
(1 +- 0.08) * 10
-1
Arg87
Ala
(3.4 +- 1) * 10
-1
(9 +- 4) * 10
-3
(2.9 +- 2) * 10
-3
Lys
(1.4 +- 0.07) * 10
-1
3.8 +- 0.4
(5 +- 0.5) * 10
-1
His89
Ala
n.d.
n.d.
<10
-5
Asn
(3 +- 0.4) * 10
-4
4 +- 1.5
(1.3
+- 0.6) * 10
-3
Asp
n.d.
n.d.
<10
-5
Gln
n.d.
n.d.
<10
-5
Glu
n.d.
n.d.
<10
-5
Lys
n.d.
n.d.
<10
-5
Asn110
Ala
n.d.
n.d.
(2 +- 0.5) * 10
-2
Gln114
Ala
n.d.
n.d.
(1.8 +- 0.5) * 10
-2
Asn119
Ala
n.d.
n.d.
< 10
-5
Asp
(4.8 +- 0.4) * 10
-3
(1 +- 0.2) * 10
-1
(5 +- 1) * 10
-4
Gln
(2 +- 0.8) * 10
-3
(6.3 +- 1.8) * 10
-1
(1 +- 0.3) * 10
-3
His
n.d.
n.d.
< 10
-5
Glu127
Ala
(3.9 +- 0.5) * 10
-2
(2.8 +- 0.6) * 10
-2
(1.1 +- 0.4) * 10
-3
Asp
(8.3 +- 2) * 10
-1
(9.9 +- 3) * 10
-2
(8.2 +- 4) * 10
-2
Gln
(1.4 +- 0.3) * 10
-2
(2.8 +- 0.9) * 10
-1
(4 +- 2) * 10
-3
Arg131
Ala
(2.4 +- 0.6) * 10
-1
(2.9 +- 2) * 10
-2
(6.9 +- 7) * 10
-2
Lys172
Ala
(9.3 +- 0.8) * 10
-2
(1.3 +- 0.2) * 10
-1
(1.2 +- 0.3) * 10
-2
Glu211
Ala
(4.1 +- 0.2) * 10
-3
2.3 +- 1
(9.4 +- 5) * 10
-3
Asp
n.d.
n.d.
1 +- 0.33
Gln
n.d.
n.d.
1 +- 0.33
REFERENCES
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