Insights on nuclease mechanism: The role of proximal ammonium group on phosphate esters cleavage

Article abstract of DOI:10.1021/ja9033236

(Figure Presented) The role of remotely located ammonium groups in the active site of many phosphate-cleaving enzymes is investigated by using model systems thus providing mechanistic hints to a still unsolved problem.

Full text of DOI:10.1021/ja9033236

Published on Web 07/23/2009
Insights on Nuclease Mechanism: The Role of Proximal Ammonium Group on
Phosphate Esters Cleavage
Renato Bonomi, Giacomo Saielli, Umberto Tonellato, Paolo Scrimin, and Fabrizio Mancin*
Dipartimento di Scienze Chimiche and CNR-ITM, UniVersit a` di PadoVa, Via Marzolo 1, I-35131 PadoVa, Italy
Received April 24, 2009; E-mail: fabrizio.mancin@unipd.it
n
n
Nucleases, enzymes hydrolyzing phosphodiester bonds in nucleic
acids, play a fundamental role in the processing of genetic information.
Table 1. Ligand (pK ) and Zn(II) Complexes (pK
a
) Deprotonation
) for 1-3 As
Constants and Zn(II) Complexation Constants (log K
Obtained from Potentiometric Titrations (NaCl 0.1 M, 25 °C, Errors Are
f
18
Moreover, they achieve record-level rate accelerations (up to 10 ) in the
within 5%)
1,2
cleavage of one of the most stable bonds in nature. As in most cases of
enzymatic catalysis, mechanisms of action of nucleases are still not fully
understood and their better comprehension, also obtained through model
studies, could give important information on chemical reactivity. A crucial
role in most nucleases is played by at least one metal ion (Mg(II), Ca(II),
and Zn(II)) present in the active site, which activates the fissile phosphate
group toward nucleophilic attack and favors the deprotonation of a
coordinated water molecule or alcoholic hydroxyl (Ser) to provide a
3
+
2+
H
n
L
[(L)Zn]
1
2
3
4
1
a
2
3
complex
pK
pK
pK
pK
log K
f
pK
pK
a
pK
a
a
1
-
9.09
10.72
7.72
7.13
7.23
5.33
5.36
5.49
1.95
<2
6.68
6.08
6.00
7.94
7.64
7.86
9.96
8.07
8.16
-
b
b
2
9.44
c
d
3
<2
-
a
b
c
d
Data from ref 5b. Ammonium group. NaClO 0.1 M. Alcoholic
4
group.
1,2
reactive nuclophile. However, fundamental contributions are also
provided by other functional groups present in the active site. In several
metallonucleases a key residue is a highly conserved Lys group located
introduced to mimic the effects of the ammonium group on the acidity of
the alcoholic residue but without concomitant electrostatic effects due to
the positive charge. Although Zn(II) is not the most diffused divalent ion
in nucleases as compared to Mg(II) and Ca(II), it allows realization of
stable complexes with a well-defined geometry that allow us to draw sound
conclusions from their study.
Potentiometric titrations confirmed the similarity of the pK
alcoholic groups of 2 and 3. Table 1 reports for the complex formation
of the metal-bound species (pKn
constants (log K ) and the pK ). The
2
just behind the water molecule best placed to attack the phosphate. Early
hypotheses suggested that the positively charged Lys ammonium group
2,3
could stabilize the developing negative charge on the transition state.
More recently, it has been proposed that the role of this group may be
that of cooperating with the metal ion in helping the deprotonation of the
a
of the two
3
attacking water molecule. An unanswered question that arises when these
two mechanisms are considered concerns the effect of the pK
a
decrease
f
a
a
of the attacking nucleophile on its reactivity. Undoubtedly metal coordina-
tion increases the amount of nucleophile available at physiological pH,
but what about its reactivity? And is further activation by external charged
centers needed, or could it be detrimental for reactivity?
ionization behavior of the complexes is quite complex. Previous investiga-
tion with 1 lead to attributing the two deprotonation events observed
1
respectively to a metal-bound water molecule (pK
group (pK
a
) and to the alcoholic
2
5b
a
). In 2, as proposed for the Lys residue in enzymes, the
Measuring ꢀnuc values of nucleophiles toward metal-activated phos-
phates is hampered by the difficulty in dissecting this contribution to
activity from other ones. We reasoned that, in the case of metal-bound
presence of the ammonium strongly affects the acidity of the neighboring
2
metal-bound alcoholic hydroxyl, lowering the pK
a
by ∼2 units. Likely,
4
such increased acidity is due to intramolecular H-bonding or electrostatic
stabilization of the deprotonated alcoholic residue. The acidity of the
alcoholic group in 3 is close to that for 2, confirming the effects of the
alkoxide nucleophiles, such as our previously studied and highly reactive
5a,b
Zn(II) complex 1 (Chart 1),
chemical modification of the alcoholic
arm could allow modulation of its acidity and evaluation of its effect on
reactivity. The high hydrolytic activity of complexes based on the bis(2-
amino-pyridinyl-6-methyl)amine (BAPA) should allow easy investiga-
tion of the substituent effect. Thus, we designed 2, where an ammonium
group is located behind the active nucleophile similarly to the Lys group
3
CF group on the alcoholic residue are similar to those of the ammonium.
The possibility of 2 to mimic a nuclease active site is also confirmed
by DFT calculation on its complex with the DNA model substrate bis-
p-nitrophenyl phosphate (BNP, Chart 1). The minimized structure (Figure
1 (left), details in Supporting Information (SI)) reveals an extended network
of intracomplex interactions. Besides the expected intramolecular H-bonds
between the pyridine amino groups and the phosphate oxygen bound to
5,6
3
in nucleases, and 3, where the electron-withdrawing CF group is
2+
+
Chart 1. Complexes and Substrates Used (for Each Substrate, the
Most Accepted Mechanism of Its Metal Ion-Promoted Cleavage Is
Reported)
3
Zn , two other relevant H-bonds, both from the NH group, are detected.
Figure 1. (Left) Solution-phase (PCM model) optimized structure of 2-BNP in
its monodeprotonated form (H-bonds evidenced by dashed red lines; colors: Zn,
gray; P, yellow; C, cyan; O, red; N, blue; H, white). (Right) pH dependence of
2+
second-order rate constant for the reaction between BNP and Zn complexes 2
-2
(
O) and 3 (b) at 25 °C ([buffer] ) 5.0 × 10 M).
1
1278 9 J. AM. CHEM. SOC. 2009, 131, 11278–11279
10.1021/ja9033236 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
a
Table 2. Kinetic pK
a
Values (pKn
a
) and Second-Order Rate
values obtained and general acid catalysis by the ammonium group is
excluded by the skie effect observed, stabilization of the developing
negative charge in the transition state by electrostatic or H-bonding
interaction with the ammonium group must be the key point.
In summary, the ammonium group in 2 produces several effects, and
not all positive, on the reactivity toward phosphate diesters. First, as
proposed for the Lys residue in enzymes, it helps the formation of the
2
+
Constants (kSUB) for Reaction of Monodeprotonated Zn
Complexes with BNP, PNPA, and HPNP
b
k
BNP
-1 -1
k
(M
PNPA
-1 -1
k
(M
HPNP
-1 -1
1
a
2
complex
pK
pK
a
(M
s
)
s
)
s )
c
1
2
3
7.9
7.3
7.7
10.2
8.4
8.6
0.097
0.022
0.0015
1.20
0.26
0.34
0.26
0.70
0.22
a
alkoxyde nucleophile by decreasing its pK by 2 orders of magnitude.
a
b
-2
From the fitting of BNP profiles. 25 °C, [buffer] ) 5.0 × 10 M,
Thus, the maximum reactivity pH is shifted closer to physiological values.
The important point that emerges from the results reported here, and that
is apparently underestimated when enzyme mechanisms are discussed, is
c
errors are within 10%. Data from ref 5b.
The first points to the alkoxyde group, and the second to the phosphate
peripheral oxygen not bound to the metal ion. The presence of these two
H-bonds indicates that the ammonium group can, in principle, participate
in the reaction playing both roles proposed for the Lys residue in enzymes:
assistance to nucleophile deprotonation and transition state stabilization.
Figure 1 (right) reports the pH dependence of the apparent second-
order rate constants for the cleavage of BNP in the presence of 2 and 3.
The profiles are bell-shaped, as previously reported for 1, indicating that
a
that this pK benefit is heavily paid in terms of reactivity loss as
demonstrated by the 65-fold lower reactivity of 3 with respect to 1. The
second effect of the ammonium group, again in line with the proposed
enzymatic mechanism, is the increased activity of the system, due to the
electrostatic effects exerted by its positive charge in stabilizing the reaction
10
transition state. In the model system studied here, electrostatic stabiliza-
tion brings about a substantial benefit with HPNP, producing the most
2+
11
active monometallic Zn complex toward this substrate so far reported.
However with BNP this effect is not strong enough to compensate for
the reactivity decrease due to nucleophile deactivation. For the Lys groups
in enzymes, if the two roles proposed, i.e., decreased basicity of the
nucleophile and stronger transition state stabilization, were mutually
exclusive, the second should be preferred since it is the only one producing
a net positive effect. However, in a less polar environment such as the
5,6b
the reactive species are the monodeprotonated complexes. The reactivity
maximum is reached at ∼pH 8 for both Zn complexes which is in line
a
with the pK values of the metal-bound species (maximum activity at pH
5
9
was, on the contrary, observed for 1). The pH profiles were fitted with
a kinetic model involving two deprotonation equilibria for the metal
complex (eq 1, SI). The pK values obtained are in good agreement with
a
those determined from potentiometric titrations. The second-order rate
constants for the reaction of BNP with the monodeprotonated complexes
are reported in Table 2. Both 2 and 3 are less reactive than parent 1, but
12
active site of the enzymes, electrostatic interaction can be much stronger.
If this were the case, the combined effect of both contributions could lead
to a substantial reactivity gain at physiological pH.
2
is sensibly more reactive than 3. Such differences are not related to
Supporting Information Available: Cartesian coordinates, synthesis
of the ligands, and kinetic details. This material is available free of charge via
the Internet at http://pubs.acs.org.
different substrate affinities. In fact, competitive inhibition experiments
carried out with dimethyl phosphate (DMP) yield almost identical binding
constants: 72, 75, and 78 M- respectively for 1, 2, and 3. Finally, the
1
References
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2
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2
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7a
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(
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(
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(
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a
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(
6) See also: (a) Feng, G. Q.; Mareque-Rivas, J. C.; de Rosales, R. T. M.;
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8
with sole nucleophilic catalysis (Chart 1); hence the reactivity toward
(
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3, 4329–4336. (b) Piatek, A. M.; Gray, M.; Anslyn, E. V. J. Am. Chem.
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8b
this substrate yields information on the nucleophilicity of the alkoxide.
The second-order rate constants for the monodeprotonated complexes
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(
8) (a) Berreau, L. M. AdV. Phys. Org. Chem. 2006, 41, 79–181. (b) Koike,
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(
(
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5
39–548, and references therein.
HPNP reaction with metal complexes is an intramolecular transesteri-
fication where the nucleophile is the substrate’s hydroxyl groups (Chart
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(
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references therein.
(
6,9
state. Note that, with this substrate (Table 2), the presence of the
ammonium group leads to the larger reactivity of 2 compared to 1 and 3.
Since ground state effects can be ruled out based on the DMP binding
JA9033236
J. AM. CHEM. SOC. 9 VOL. 131, NO. 32, 2009 11279