Standard Free Energy for the Hydrolysis of Adenylylated T4 DNA
Ligase and the Apparent pKa of Lysine 159*
Abolfazl
Arabshahi and
Perry A.
Frey
From the Institute for Enzyme Research, The Graduate School, and
Department of Biochemistry, College of Agricultural and Life Sciences,
University of Wisconsin, Madison, Wisconsin 53705
 |
ABSTRACT |
Equilibrium constants for the adenylylation of T4
DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a
pKa of 8.4 ± 0.1. The simplest interpretation
is that the apparent pKa is that of the 6-amino
group of the AMP-accepting residue Lys159. Based on the pH
dependence of the equilibrium constants, the value at pH 7.0 is 0.0213 at 25 °C, corresponding to
G'o = +2.3
kcal mol
1. From this value and the standard free energy
change of -10.9 kcal mol
1 for the hydrolysis of ATP to
AMP and PPi, we calculate that
G'o for the hydrolysis of the adenylyl-DNA
ligase is -13.2 kcal mol
1. The presence of conserved
basic amino acid residues in the catalytic domain, which are proximal
to the active site in the homologous catalytic domain of T7 DNA ligase,
suggests that the pKa of Lys159 is
perturbed downward by the electrostatic effects of nearby positively
charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of
lysine and the high energy of the
,
-phosphoanhydride linkage in
ATP significantly facilitate adenylylation of the enzyme.
 |
INTRODUCTION |
DNA ligases catalyze phosphodiester-bond formation between the
adjacent 5'-phosphate and 3'-hydroxyl ends of the nicked DNA chains (1,
2). DNA ligases were first identified in 1967 (3-8) and shown to play
essential roles in DNA replication, repair, and recombination.
Recently, interest in DNA ligases has been expanded by new information
regarding the connections between human cancers and DNA repair systems
(9, 10). The two types of DNA ligases are ATP-dependent and
NAD+-dependent, respectively.
ATP-dependent ligases are from eukaryotic cells, certain
prokaryotes, and bacteriophages of the T series; NAD+-dependent enzymes are from other
prokaryotic cells. DNA ligases require a divalent cation for activity,
and the optimum concentration of Mg2+ for the T4 DNA ligase
is 10 mM (2). Molecular weights of DNA ligases vary
considerably, from 41 kDa in T7 to more than 100 kDa for the mammalian
DNA ligases. The T4 DNA ligase is a single polypeptide with the
molecular weight of 68,000 (11). The crystal structure of T4 DNA ligase
has not been determined; however, that of T7 DNA ligase has been solved
at 2.6 Å (10).
DNA ligation proceeds in three reversible steps by a ping pong kinetic
mechanism (2, 12) as shown in Eqs. 1a-1c for ATP-dependent ligases.
|
(Eq. 1a)
|
|
(Eq. 1b)
|
|
(Eq. 1c)
|
DNA ligase reacts with ATP in the first step to displace
PPi and generate an AMP enzyme (adenylyl enzyme) complex in
which the AMP is linked to the 6-amino group of a lysine residue of the
enzyme through a phosphoramide bond (13). The AMP enzyme then binds
nicked DNA and transfers the AMP group to the 5'-phosphoryl group in
the nick. The resulting adenosine-5'-pyrophosphoryl moiety in the nick
activates the phosphate group at the 5' terminus of the DNA by
supplying AMP as a good leaving group. The final step is a nucleophilic
attack by the 3'-hydroxyl group on this activated phosphorus atom to
displace AMP and form the 3'-5'-phosphodiester bound.
The second and the third steps (Eqs. 1b and 1c) are thermodynamically
spontaneous reactions (i.e. 
G'°). However,
the adenosine-5'-phosphoramidate formed in the first step is expected
to be a high energy intermediate. The standard free energy change for
the hydrolysis of E-Lys-NH-AMP (nucleoside-5'-phosphoramidate) has not been known; however, it could
be calculated from the equilibrium constant for adenylylation of the
enzyme at pH 7.0 and the standard free energy change of
10.9 kcal
mol
1 for the hydrolysis of ATP to AMP and PPi
(14). In this study, we report the equilibrium constants for the
adenylylation of T4 DNA ligase at ten different pH values, the apparent
pKa for dissociation of the protonated lysine
residue of the T4 DNA ligase, and the standard free energy change for
the hydrolysis of adenylylated T4 DNA ligase.
 |
EXPERIMENTAL PROCEDURES |
Materials--
ATP,
CHES,1 HEPES, dithiothreitol,
ovalbumin, and DNase were purchased from Sigma. The following were
obtained from the vendors indicated: Mg acetate from Aldrich; pET21
vector from Novagen; YM30 filtration membrane from Amicon;
[3H]ATP from ARC; Ultrafree-MC 30K filter unit from
Millipore; and Bio-Safe II from Research Products International Corp.
Preparation of DNA Ligase--
T4 DNA ligase was purified from
Escherichia coli BL21(DE3) transformed with plasmid pEAW128.
To construct pEAW128, the gene encoding T4 DNA ligase was inserted into
pET21A (Novagen) in two PCR fragments. Genomic DNA of E. coli strain BNN67, in which the T4 ligase gene is inserted as a
bacteriophage lysogen (15), was used as the template for both PCR
reactions. Genomic DNA was prepared using a published procedure (16).
The 256 base pairs of the T4 ligase gene defining the N terminus of the
protein from the initiation codon to the MfeI site were
inserted into pET21A, replacing sequences from the NdeI to
the BamHI sites. The 5'-PCR primer corresponded to bases
1-18 of the T4 ligase gene, with 5'-CTCGAGCCAT added to the 5' end to
provide a clamp and an NdeI site. The 3'-PCR primer
corresponded to bases 261-247 of the T4 ligase gene, with 5'-CGGGATCC
added to the 5' end to provide a BamHI site and clamp. With
the first fragment inserted, the modified pET21A was digested at
MfeI and HindIII, and the remaining base pairs of
the gene were inserted. The 5'-PCR primer for this second fragment
corresponded to bases 254-265 of the T4 ligase gene. The 3'-primer
corresponded to bases 1460-1455 of the T4 ligase gene, with
5'-CCCAAGCTTTCA added to provide a clamp and HindIII site.
DNA ligase was purified by a modification of the procedure of Davis
et al. (15). Cells were disintegrated by addition of DNase
and then sonicated for 4 min in 1 min bursts. At the early stage of
purification two ammonium sulfate fractionations were performed. The
last step of the procedure, hydroxylapatite chromatography, was omitted
because the enzyme was essentially pure as indicated by gel
electrophoresis. Purified enzyme was concentrated in an Amicon
ultrafiltration device using the YM30 filtration membrane.
Equilibrium Constant for Adenylylation of DNA Ligase--
The
equilibrium constants (KpHx) for the
reaction of DNA ligase and [3H]ATP·Mg to produce the
covalent AMP enzyme and pyrophosphate according to Eq. 2 were measured
at ten different pH values.
|
(Eq. 2)
|
The buffers used for the different pH values were as follows:
CHES for pH values 10.0, 9.5, 9.0; TAPS for pH values 8.5, 8.4, 8.2;
and HEPES for pH values 8.0, 7.8, 7.5, 6.8. Each reaction mixture (50 µl) consisted initially of 50 mM buffer, 10 mM dithiothreitol, 0.0025-1 mM of
Mg-[3H]ATP, 1 mM Mg-acetate, and 0.012 mM DNA ligase. The reactions proceeded for 20 min at
25 °C. The reactions were stopped by addition of 550 µl of 15 mM EDTA. The solutions were filtered through Ultrafree-MC (30-kDa cutoff) filter units, which separated the enzyme from the small
molecules. Ultrafree-MC filter units with a low binding regenerated
cellulose membrane are essentially a 1.5-ml polypropylene conical
microcentrifuge tube into which a 400-µl chamber with the membrane
forming its bottom is inserted. An aliquot of the reaction mixture (300 µl) was transferred from the reaction vessel into the upper chamber
of the filter unit, and the tube was subjected to centrifugation at
2,000 × g for 30 min in a microcentrifuge (MicroV;
Denver Instrument Company). The sample was completely centrifuged
through the filter. The upper chamber was then transferred to a fresh
microcentrifuge tube. An aliquot of 50 mM buffer (300 µl)
was added to the concentrate to initiate the washing, and the tube was
centrifuged at 2,000 × g for 20 min in a
microcentrifuge. This procedure was repeated three times. After the
final wash, the upper chamber containing the concentrate was
transferred into a scintillation vial, then 400 µl of 10% (w/v) SDS
was added to the upper chamber inside the vial. After 1 h, 600 µl of the 50 mM buffer and 15 ml of Bio-Safe II counting
mixture were added into the scintillation vial, which was then counted
for [3H]AMP ligase. Nonspecific binding to the filter was
determined in the same way using samples in which chicken ovalbumin was
substituted for DNA ligase. To confirm that the reactions had reached
equilibrium in 20 min, two of the reactions (0.04 and 0.1 mM ATP at pH 8.5) were carried out for three different
reaction periods (20, 30, and 40 min). The results showed no
significant differences in the production of 3H-labeled
ligase after 20 min.
Apparent equilibrium constants (KpHx)
for adenylylation of DNA ligase (Eq. 2) at different pH values were
calculated by using following equation.
|
(Eq. 3)
|
In a given measurement of KpHx,
the values of [E-AMP] were obtained as ligase-bound
tritium at each concentration of Mg-[3H]ATP, and
Eo was the maximum amount of ligase-bound tritium.
Evaluation of the Apparent pKa for
Lys159--
The pKa for the
dissociation of Lys159 in DNA ligase refers to the process
of Eq. 4.
|
(Eq. 4)
|
Assuming that only the unprotonated form of Lys159
reacts with Mg·ATP, the apparent dissociation constant for
Lys159, Ka, is given by Eq. 5, where
KpHx is the apparent equilibrium
|
(Eq. 5)
|
constant at pH = x, and
Keq is the pH-independent overall equilibrium constant.
 |
RESULTS AND DISCUSSION |
Equilibrium Constants for the Formation of
E-Lys159-NH-AMP--
The covalent intermediate is produced
in the reaction of DNA ligase with ATP according to Eq. 1a. The
apparent equilibrium constant at a given pH = x,
KpHx, can be measured by use of
radiolabeled ATP as described under "Experimental Procedures." The
equilibrium constants for adenylylation of DNA ligase were determined
at ten different pH values and have been plotted against pH in Fig.
1.

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Fig. 1.
KpHx
versus pH for adenylation of DNA ligase. The
apparent equilibrium constants, KpHx,
were measured as described under "Experimental Procedures." The
results were then plotted as KpHx
versus pH. Then from fitting the data to Eq. 5 the line was
computed as well as the apparent pKa for
Lys159. The pKa value resulting from
this fitting procedure was 8.40 ± 0.09.
|
|
The Apparent pKa for Lys159 of T4 DNA
Ligase--
The data for apparent equilibrium constants,
KpHx were fitted to Eq. 5 to obtain the
apparent Ka. The value of the apparent
pKa of Lys159 is 8.40 ± 0.09 at
25 °C. This value is 2.1 units less than the normal
pKa for dissociation of the protonated 6-amino group
of a lysine residue, which is 10.5 (17). The simplest interpretation of
the results in Fig. 1 is that the apparent pKa observed is that for the dissociation of the 6-ammonium group of
Lys159.
A value of pKa 2.1 units lower than usual can be
rationalized on the basis of the existence of a net positive charge in
the active site of the free enzyme. Although the structure of the T4
DNA ligase is not available, the crystal structure of T7 DNA ligase has
been solved at 2.6 Å resolution (10). Aside from the differences in
molecular weights of the two enzymes, the amino acid sequence alignment
of T4 and T7 DNA ligases revealed the existence of conserved motifs in
the catalytic domain (18). The crystal structure of T7 DNA ligase
complexed with ATP shows an excess of positive charges surrounding the
nucleotide molecule (19), and almost all lysine and arginine residues
at the active site of T7 and T4 DNA ligase are conserved (18). These
residues include, in addition to the AMP-accepting Lys159
(20), Lys375, Lys277, Arg182, and
Arg164 of T4 DNA ligase. A lowered value of
pKa for Lys159 in the active site of the
T4 DNA ligase, from 10.5 to 8.4, could well be caused by electrostatic
destabilization of the lysine-6-ammonium group by the nearby positively
charged, basic amino acids. The positive electrostatic field would
favor proton dissociation and a lowered pKa. This
effect might not be confined to Lys159 and would perturb
the acid dissociation constants of the other basic residues as well, it
is only the effect on the pKa of Lys159
that would be detected by our chemical test of adenylylating the enzyme.
Standard Free Energy Change for Hydrolysis of Adenylylated DNA
Ligase--
The recommendation of the International Union of Pure and
Applied Chemistry is that
G'o refers to total
concentrations of the ionic species of each component of the reaction
at pH 7.0, 25 °C, and 1 mM free Mg2+ (21).
In this study, standard free energy changes follow this recommendation.
The standard free energy change for hydrolysis of adenylylated DNA
ligase,
G'o, can be calculated from the free
energies of the reaction studied here, Eq. 1a, and that for the
hydrolysis of ATP to AMP and PPi (22). To determine the
G'o, the value for
KpH7 at pH = 7 for the adenylylation of DNA
ligase was calculated using Eq. 5 and found to be
KpH7 = 0.0213. Then the
G'o for Eq. 1a was calculated as +2.3 kcal
mol
1 from
G'o =
RT
ln KpH7. The standard free energy change
(
G'o °) for hydrolysis of adenylylated DNA
ligase, Eq. 8, can be calculated as the sum of Eqs. 6 and 7 as shown in
Table I. The value of
G'o is
13.2 kcal mol
1, which
indicates that E-Lys-NH-AMP is a high-energy compound. As we
expected (14) the standard free energy of hydrolysis of adenylylated
DNA ligase is in the same range as the value of
G'o for the hydrolysis of UMP-imidazolide to
UMP and imidazole, which is
14.7 kcal mol
1 (22).
Although adenylyl-DNA ligase is high energy, it is not so high as to be
inaccessible by cleavage of the phosphoanhydride bond linking
P
and P
in Mg·ATP, which is 2.3 kcal
mol
1 lower in energy when expressed as the standard free
energy of hydrolysis. The difference can be overcome by the millimolar
concentration of Mg·ATP in a cell and the very favorable free energy
changes for the coupled reactions Eqs. 1b and 1c. The low apparent
pKa of Lys159 is a significant factor in
bringing the value of
G'o near enough to that
of Mg·ATP for cleavage to AMP and Mg·PPi. If the
apparent pKa of Lys159 had been normal
for the 6-amino group (10.5),
G'o for the
hydrolysis of adenylyl-DNA ligase would have been
16.1 kcal
mol
1, a value that would make the formation of the
adenylyl-DNA ligase intermediate very difficult. Therefore, it seems
that electrostatic pKa perturbation of
Lys159 at the active site of DNA ligase is significant in
facilitating the action of this enzyme.
Another factor favoring the adenylylation of DNA ligase is the energy
of the
,
-phosphoanhydride linkage in ATP. The standard free
energy of hydrolysis has recently been found to be
10.9 kcal
mol
1 (14), which is significantly more negative than the
7.8 kcal mol
1 for hydrolysis of the
,
-phosphoanhydride linkage (21). The combined effects of the high
energy
,
-phosphoanhydride linkage in ATP and the low value of
pKa for Lys159 in T4 DNA ligase
facilitate the formation of the intermediate AMP ligase.
Another high energy intermediate in the action of DNA ligase is the
adenylylated DNA produced by transfer of the AMP-group from the
adenylyl-DNA ligase to the 5'-P of nicked DNA. The resulting phosphoanhydride linkage is a P1,P2-dialkyl
diphosphate, and as such can be expected to display a similar value of
G'° to that for the hydrolysis of UDP-glucose to UMP
and glucose-1-phosphate, which is
10.3 kcal mol
1 (14).
This is 2.9 kcal mol
1 less negative than
G'° for the hydrolysis of adenylyl-DNA ligase, so that
the transfer of the AMP group from the adenylylated enzyme to nicked
DNA appears to be spontaneous.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Elizabeth A. Wood
and Professor Michael M. Cox, who constructed and supplied the strain
used to produce DNA ligase for this research.
 |
FOOTNOTES |
*
Supported by Grant GM 30480 from the National Institute of
General Medical Sciences.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 608-262-0055;
Fax: 608-265-2904.
 |
ABBREVIATIONS |
The abbreviations used are:
CHES, 2-(N-cyclohexylamino)ethanesulfonic acid;
DNase, deoxyribonuclease;
TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic
acid;
PCR, polymerase chain reaction.
 |
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