From the
¶Departments of Chemistry and Biochemistry
and **Biological Science and the
Institute of Molecular Biophysics, Florida
State University, Tallahassee, Florida 32306-4380
Received for publication, December 18, 2002 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Phosphagen kinases are among the most intensively studied by classic enzymology (3) and constitute paradigms for the fundamentals of catalysis of multisubstrate reactions. Pre-ordering and alignment of substrates likely contribute more to the catalysis of bimolecular reactions than the better characterized unimolecular reactions, but there is little consensus on its importance relative to acid-base chemistry, strain, and other catalytic effects (46). One challenge was a lack of high resolution structures of multisubstrate enzyme complexes in which the alignment of two substrates could be viewed without perturbation or constraint, an obstacle overcome first with the structure of arginine kinase (7).
Several phosphagen kinase structures have become available recently, including creatine kinases from chicken muscle mitochondria (8), rabbit muscle (9), chicken brain (10), human mitochondria (ubiquitous) (11), and arginine kinase from horseshoe crab (12), all structures determined in the open, inactive configuration. The structure of arginine kinase was also determined as a transition state complex (TSAC1; Mg2+-ADP, nitrate, arginine) (7) in a closed conformation with ordered active site loops. This structure prompted re-examination of the catalytic mechanism here and elsewhere (see below). The recent structure of creatine kinase from Torpedo californica revealed one subunit in a binary Mg-ADP complex, the other as TSAC, confirming that the transition state structures of creatine and arginine kinases were very similar (13).
Prior to these structures, biochemical kinetics, chemical modification, and site-directed mutagenesis led to a proposed mechanism of in-line phosphoryl transfer involving abstraction of a guanidinyl proton as an early step, catalyzed, it was long thought, by a histidine (14). The arginine kinase TSA structure, in fact, showed only two residues, Glu-225 and Glu-314, in contact with the phosphorylated guanidinium nitrogen (Fig. 1). If the pKs of the glutamates were matched to the reactant arginine for efficient isoergonic proton transfer, then it implied that proton abstraction would not be initial but subsequent to or coincident with addition of the new NP bond when the nitrogen would have lower pK (7). Consistent with this, the glutamates were positioned for interactions favoring a tetrahedral reactive nitrogen. The TSAC structure, now refined to 1.2 Å resolution (15), suggested another potential role of the glutamates. The reactants were all aligned precisely within 3° of optimal for in-line transfer. This raised interesting questions about the relative roles of acid-base chemistry and substrate pre-ordering in the catalysis of this multisubstrate reaction and the roles in each played by these two glutamates.
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Glu-225 is highly conserved, and mutagenesis soon suggested that the corresponding residues in creatine kinase were critical for (acid-base) catalysis: Glu-227 and Glu-232, respectively, in the human mitochondrial and muscle enzymes (16, 17). Glu-314 of arginine kinase is in a poorly conserved region, aligned with a valine in most alignments to creatine kinase sequences (18). However, prior to the recent T. californica creatine kinase structure (13), it could also be aligned with a neighboring aspartate, conserving a carboxylate throughout the family (7). Mutagenesis of Asp-326 in human muscle creatine kinase indicated an essential role, perhaps in substrate alignment (17). Here we report mutagenesis and structural studies that suggest a different interpretation of catalysis and the roles of these amino acids.
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EXPERIMENTAL PROCEDURES |
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Original CloneTwo variants of an arginine kinase expression
system were used, both derived from a pET 22b (Novagen) plasmid clone
(19) that contains a
selectable ampicillin resistance gene. Escherichia coli strain
BL21(DE3)pLysS was transformed for expression of protein, whereas strain
DH5 was used to produce plasmid DNA for mutagenesis. The first
expression system was the same as used in the original structure determination
(7,
20) and is termed
AKorig. Sequencing, prompted by the structure, revealed
PCR-generated nucleotide misin-corporations leading to four amino acid
changes, E103Q, D112G, G116A, and K351R. These were reverted back to the
native sequence using the QuikChange mutagenesis kit (Stratagene)
consecutively, in the order G112D, Q103E, A116G, R351K, using plasmid from the
prior round of mutagenesis as the template for the next round. Sequences were
confirmed using an ABI model 3100 DNA sequencer. The final native clone has
been designated as reverted wild type AKrev.
Mutagenesis was carried out using both AKorig and AKrev templates and the QuikChange kit. Sequences of constructs were confirmed by sequencing from both ends of the gene and with internal primers.
Expression and Protein Purification from
AKorigSmall cultures of E.
coli containing the plasmid were grown overnight at 37 °C in Luria
broth with 100 µg/ml ampicillin. One-liter cultures were inoculated with 20
ml of this overnight culture, grown to mid log phase (optical density
A600 = 0.5), and protein expression was induced with a
final concentration of 1 mM
isopropyl-1-thio--D-galactopyranoside. Cells were harvested
at 56 h postinduction and lysed in a French pressure cell. Protein was
expressed as inclusion bodies, unfolded in 8 M urea, and refolded
by sequential dialysis against decreasing concentrations of urea. Enzyme from
this system was purified by previously reported chromatographic methods
(20).
Expression and Protein Purification from Wild Type AKrevThe same procedures were used for E. coli culture and induction. However, protein was mostly in the soluble fraction after lysis. The properly folded and active protein was put directly onto a DEAE anion exchange column after dialysis against running buffer (10 mM Tris, pH 8, 10 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.02% NaN3) and eluted with a 0200 mM KCl gradient. This was followed by size exclusion chromatography on a S100 Sephacryl column. Chromatography was carried out using an ÄKTA FPLC (Amersham Biosciences).
ConcentrationProtein was concentrated for crystallization using Centricon-10 microconcentrators or an Amicon cell with 10-kDa molecular mass cutoff YM-10 membranes. Protein concentration was determined spectrophotometrically at A280, using an extinction coefficient of 0.76 ml·mg1·cm1. For enzyme dialyzed against TSA components, the concentration was measured with the BioRad protein reagent microassay and uncomplexed arginine kinase as a standard.
Enzyme KineticsPhosphoryl transfer was assayed in the reverse direction by measurement of the increase in absorbance at 340 nm due to production of NADPH resulting from linkage of ATP production to the hexokinase and glucose-6-phosphate dehydrogenase reactions (21). Note, in contrast to creatine kinase, the substrate binding constants for arginine and Mg-ATP for the forward reaction are similar to those for the reverse direction (phosphoarginine and Mg-ADP) (22, 23). Kinetics assays were set up in a 6 x 6 matrix of ADP versus phosphoarginine concentrations, with phosphoarginine between 0.2 and 3.2 mM and ADP between 0.03 and 0.96 mM. Concentrations of ADP in assay stocks were verified by UV absorbance, whereas phosphoarginine concentrations were determined by a spectrophotometric enzymatic assay using highly purified arginine kinase. Assays were conducted in triplicate at 25 °C using a Varian Cary 3E UV-VIS spectrophotometer. Data were analyzed using SigmaPlot (SPSS, Inc.).
CrystallizationAKorig protein at 1020
mg/ml was dialyzed against transition state analog components
(7) and crystallized by vapor
diffusion at 4 °C. Crystals of E314D measuring 1.0 x 0.3
x 0.3 mm3 were obtained through both macro-seeding
(24) and de novo
set-ups. For seeding, a 10-µl drop comprised of 5 µl of 20 mg/ml protein
plus 5 µl of 26% PEG6000, 0.025 M HEPES, pH 7.5, 0.05
M MgCl2 was aliquoted onto a coverslip and then seeded
with a piece of an AKorig crystal (grown under the same
conditions). The drop was then equilibrated by vapor diffusion against 26%
PEG6000. For de novo crystallization of E314D, the drop contained 5
µl of 20 mg/ml protein plus 5 µl of 32% PEG6000, 0.025 M
HEPES, pH 7.5, and 0.05 M MgCl2 for a drop concentration
of 16% PEG6000. It was equilibrated by vapor diffusion against 28% PEG6000.
The same conditions were used for de novo crystallization of E225Q,
except that drops were made from 2 µl of
24 mg/ml protein solution
plus 2 µl of 26% PEG6000. De novo crystals of E225Q were not of
sufficient quality but were used for microseeding
(24) identical conditions.
Data CollectionDiffraction data were collected with an R-Axis II image plate detector (Rigaku/Molecular Structure Corporation) and a rotating anode x-ray source at our in-house facility. E314D/TSA and E225Q/TSA data were collected at 100 K using 25% PEG6000 + 20% glycerol as the cryoprotectant.
Structure DeterminationThe Denzo/Scalepack/HKL suite (25) was used for data processing. Rotation and translation searches were carried out with GLRF (26), using TSAC arginine kinase as a model. Manual rebuilding using the program "O" (27) was alternated with atomic refinement using the CNS package (28).
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RESULTS |
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KineticsKinetic values from the 6 x 6 matrix assay
yielded Km, KS,
Vmax, and kcat values for the wild
type mutants (Table I). Here,
Km is defined as the ternary steady-state
constant (a measure of binding the second substrate in this random-order rapid
equilibrium reaction (22)),
whereas KS, denoted as KiA by some
authors, is the "initial" binary steady-state constant. Kinetic
plots for the wild type mutants were consistent with the random order bi-bi
mechanism (data not shown). Individual mutations to Asp and Gln at positions
E225 and E314 and the double mutation E225Q/E314Q had little impact on the
Km and KS values for ADP and
phosphoarginine yet reduced the catalytic activity 60500-fold
(Table I). Substrate binding
synergy, one substrate facilitating the binding of the second and
characterized by =
Km/KS < 1, is similar to
that of chicken cardiac mitochondrial and rabbit muscle creatine kinases
(29,
30). There is a partial loss
of synergy with mutation at Glu-225, but the change is not always
experimentally significant and involves different effects upon
Km and KS for different
mutants. Overall, catalytic activity is affected much more than substrate
binding.
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The most surprising result came from a multisite mutant that included the non-conservative mutation E314V. This chimeric construct, containing several substitutions of creatine kinase residues into arginine kinase, was part of an on-going study of substrate specificity. It became relevant to this study with the unexpected finding of only 2-fold loss of activity despite a non-conservative change at one of the putative catalytic bases. The non-conservative mutation at the other glutamate, E225A, showed 3,000-fold reduction in activity relative to wild type, precluding full kinetic analysis but confirming that this residue also is not absolutely essential for catalysis.
E314D/TSA StructureThe crystal of E314D was nearly
isomorphous with wild type (Table
II), and its structure was solved by molecular replacement using
the CNS package (28).
Cross-rotation and translation searches using the wild type transition state
model (7) yielded a unique
solution, rotated 1.9° from the wild type with slightly different crystal
packing. The final model includes an ADP molecule, a nitrate, one
Mg2+, a substrate arginine, and 395 water molecules. It
has R/Rfree of 0.18/0.24 and only 0.6% of ,
angles in the generously allowed region of a Ramachandran plot (none
disallowed) (31).
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Overall, the E314D mutant structure is similar to wild type, with an
overall root mean square difference between the C atoms of the two
structures of 0.39 Å. Localized differences near the loop containing 314
include (Fig. 1C): (i)
movement of the side chain carboxylate of E314D by 0.7 Å so that
separate O
1 and
O
2 interactions with substrate arginine
N
and N
2 are
replaced by a single bridging interaction of
O
2 to both the
N
and N
2
(Fig. 1D); (ii)
movement of the loop near 314 by up to 0.9 Å
(Fig. 1C); (iii) small
changes in the alignment of the substrate analogs
(Fig. 1E and
Table III), due to movements of
the ADP phosphates, Mg2+,
NO3, and arginine by up to 0.25 Å (detailed
below); and (iv) a movement of His-315 away from the active site, 1.5 Å
at the C
atom (Fig.
1C). The movement of His-315 and the 180° flipping of
its
2 side chain torsion angle establishes a possible
2.6-Å salt bridge interaction with the terminal carboxyl group of the
substrate arginine that is not present in wild type
(Fig. 1D). It also
disrupts a water-mediated interaction to the backbone carbonyl of residue 62,
on the other flexible loop, reducing the distance from 5.9 to 3.7 Å, too
short for an intervening water but too long for a direct hydrogen bond.
However, this has little impact on the backbone structure near residue 62.
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The changes in substrate alignment arise mostly from a translation of the
nitrate by 0.25 Å away from Arg-309 and a small tilting of the
nitrate plane so that it is not so orthogonal to the direction of in-line
transfer. Although the mutated Asp-314 is a contact residue 3.7 Å from
the nitrate O1, there is not a specific interaction that causes the move, but
the mutation would change the local electrostatic environment. The move of the
nitrate is small but clear from the electron density. The ADP
-phosphate
and substrate arginine guanidinium translate roughly parallel but by a smaller
amount (<0.2 Å). The result is little change in the directions of
nucleophilic attack from the ADP O3
or
the guanidinium N
2 but distortions in the
angle of approach to the nitrate plane (that is mimicking the transferring
phosphoryl) of up to 9°.
Excluding the loop containing the E314D, the protein is essentially unchanged and the root mean square difference is nearly halved to 0.22 Å. Conserved arginines 124, 126, 229, 280, and 309 maintain unchanged interactions with substrate phosphate groups with, at most subtle, change to accommodate the slightly altered phosphate positions. The "essential" Cys-271 and its interactions are unperturbed.
E225Q/TSA StructureThe crystal was also nearly
isomorphous with wild type (Table
II), and its structure was similarly solved by molecular
replacement. The final model contained an ADP, a nitrate, an
Mg2+ ion, a substrate arginine, and 126 water molecules.
It has R/Rfree values of 0.17/0.25, and only 0.6%
of ,
angles are "generously allowed" (none disallowed)
(31)
(Table II). Overall, the
changes of E225Q compared with wild type are more modest than for E314D. The
overall root mean square difference in C
atoms between E225Q and wild
type is 0.27 Å. At the site of mutation, the backbone shift at residue
225 is small, <0.2 Å. The largest changes are once again in the side
chain of His-315, with a shift at C
of 0.5 Å
(Fig. 1D), smaller
than in E314D and not enough to disrupt the water-mediated interaction with
the carbonyl of residue 62. The closer interaction of His-315 with the
substrate carboxylate (Fig.
1D) appears to have little impact upon binding
(Table I), presumably because
it is offset by the loss of similar ionic interactions between the guanidinium
and Glu-225 as it is changed to Gln. Although there are other changes in
hydrogen bonding, the interactions of the reactive
N
2 with residues 225 and 314 that favor
transition toward a tetrahedral nitrogen appear to be unaffected
(Fig. 1D). As in
E314D, the linearity of phosphoryl transfer is perturbed
(Fig. 1D and
Table III). Positional shifts
here are in the range of 0.240.34 Å, excepting ADP
O3
, which has not moved significantly.
The largest change is a 26° rotation and tilting of the nitrate,
distorting the nucleophilic attack angles to the nitrate by as much as 22°
(Table III). Remote from the
site of mutation, changes are minor. When substrate-contact residues are
omitted, the root mean square difference between wild type and mutant
structures is 0.24 Å. The positions of "essential" Cys-271
and the conserved arginines (124, 126, 229, 280, 309) have not changed
significantly, although Arg-124 has lost one of its two hydrogen bonds to ADP
O2
.
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DISCUSSION |
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Near wild type activity (83% kcat) for a multisite mutant protein containing an E314V substitution shows that Glu-314 is not a critical catalytic base. There is now no reason to pair it with aspartates in sequence alignments (7). In fact, the recent TSA structure of T. californica creatine kinase aligns Val-325 with arginine kinase Glu-314, whereas Asp-326 is more remote from the creatine nitrogens (13). Val-325 interacts with the CH3 that is unique to creatine. It remains to be seen whether the drastic effects of mutating the aspartate in creatine kinase (17) reflect differences between creatine and arginine kinases or whether more innocuous mutations remain to be discovered as for arginine kinase Glu-314.
The 300-to-3000-fold attenuation of activity for the E225Q/D/A mutations is similar to the E314D/Q mutations at a non-essential site and less than expected for a base in the primary means of catalysis. Comparing to corresponding creatine kinase mutants, they are similar to a 500-fold attenuation in the human muscle E232D mutant (17) but differ from the 90,000-fold attenuation in the mitochondrial E227Q mutant (16) and greater attenuations with non-conservative mutations (16, 17). Retention of significant activity in the arginine kinase E225A mutant suggests that Glu-225, like Glu-314, is not absolutely essential. Arginine kinase might be unique with possible redundancy offered by the juxtaposition of a second glutamate (Glu-314). This possibility is eliminated by the double mutant E225Q/E314Q having activity similar to the E225Q single mutant (Table I). Thus, the conserved Glu-225 has a more modest role in a multifaceted catalytic mechanism than implied from earlier creatine kinase mutants (16).
The similar kinetic effects of mutations at Glu-225 and Glu-314 raise the possibility that they impact the reaction in similar ways, not necessarily involving formal proton transfer. Conservative mutations at Glu-314 or Glu-225 reduce activity somewhat without greatly affecting substrate binding (Table I). In the E225Q structure, a hydrogen bond is preserved between the substrate and Gln-225 that could contribute to catalysis, short of formal proton transfer, by lowering the pK of the reactive nitrogen, perhaps explaining the modest additional loss of activity without the hydrogen bond in the E225A mutant. Significant activity in E225A indicates that Glu-225 acts in a supplemental, not an essential, capacity. Similar kinetics in the arginine kinase E225D and human muscle creatine kinase E232D mutants (17) support a common role, but the disparity with mutations in other homologs (16, 17) indicates a susceptibility to collateral damage. Thus, the difference in E225A and E225Q kinetics could be attributed to the absence of pK perturbation, collateral damage, or perturbation of substrate alignment. The non-additivity of the kinetic effects of E225Q and E314Q in a double mutant suggests that their effects are mediated through a common means, mostly likely substrate alignment, having ruled out pK effects for Glu-314 (see above).
Conformational changes are larger in E314D than E225D and mostly involve
distortions to the loop containing 314 as it maintains an interaction with the
substrate arginine despite a shorter side chain. There is a greater loss of
activity in E225Q despite smaller overall changes to the active site,
indicating that the two may not be strongly correlated. Changes to the atoms
involved in the phosphoryl transfer may be more critical: up to 0.25 Å
for E314D and 0.34 Å for E225Q. These changes are commensurate with the
estimated errors of the structures (0.27 and 0.38 Å, respectively), and
only slightly increase distortion of the linear transfer (from 173° in
wild type to 170° in E225Q and 168° in E314D;
Table III). The angles of
nucleophilic attack at the guanidinium N2
and ADP O3
remain close to perfect in
E314D but are distorted 5 and 10°, respectively, in E225Q. Obvious from
the E225Q electron density is a change in the tilt of the nitrate as the
oxygens are moved 0.5 to 0.9 Å, leading to a 22° distortion of the
angle at which lone pairs of the guanidinium or ADP would attack the phosphate
in each direction of the reaction. Thus, precise substrate alignment may be an
important role for Glu-225.
Catalytic rate could also be enhanced by restricting substrate motion (entropy). Crystallographic "B"-factors reflect static and temporal disorder but also crystal quality. Diffraction from E314D was of comparable quality to the initial 1.9 Å wild type structure. B-factors for substrates in both wild type and E314D were below average for the protein, indicating restricted active site motion, but they were similar in wild type and mutant, indicating that motion is not the cause of reduced activity in the mutant.
In summary, near normal activity in a non-conservative mutant excludes Glu-314 from general base catalysis, whereas the structure of a conservative E314D mutation with modestly reduced activity shows that subtle perturbations to precise substrate alignment can be important. Glu-225 has a more important role, but still accessory and non-essential, perhaps through general base catalysis or substrate pre-alignment. The mutant structures show that the required precision of alignment is fine, with 514° degrees of misalignment reducing the rate by 23 orders of magnitude on its own (E314D) or in combination with acid-base effects (E225Q). This would be similar to that predicted by Koshland's orbital steering (32), although the foundations of that theory have long been questioned (33). The perturbations in substrate alignment are commensurate with the range of near-attack configurations (±15°) in more contemporary simulations that account for both entropic and enthalpic components in substrate pre-ordering (4, 5). They are also commensurate with the substrate-cofactor alignment required in isocitrate dehydrogenase (34).
Some ambiguities remain because perturbations of substrate alignment, a possible cause of reduced activity in mutants, are commensurate with the precision of available structures. The most important results are negative; dominant roles for Glu-225 and Glu-314 as catalytic bases are ruled out by new mutants retaining substantial activity, and the absence of large structural changes highlights the importance of subtle structural perturbations. Although possible, substantial differences in the mechanism of arginine and creatine kinases seem implausible, so the discrepancies between some of the homologous mutations is a reminder that loss of activity need not imply a direct role of that residue in the mechanism. Several mutations may be required in more than one homolog before finding one that is free from unpredictable collateral effects that, especially in the absence of structure, might lead to an incorrect mechanistic interpretation.
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FOOTNOTES |
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* This work was funded by National Institutes of Health Grant R01GM55837 (to
M. S. C). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Supported in part by National Science Foundation Research Training Grant
DBI96-02233.
|| On leave from the Dept. of Biophysics, Faculty of Science, Cairo
University, Cairo, Egypt.
To whom correspondence should be addressed. Tel.: 850-644-8354; Fax:
850-644-7244;
E-mail:chapman{at}sb.fsu.edu.
1 The abbreviations used are: TSAC, transition state analog complex;
AKorig, original clone of arginine kinase containing four PCR
amplification coding errors; AKrev, clone with native sequence
restored through mutagenesis; TSA, transition state analog.
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ACKNOWLEDGMENTS |
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REFERENCES |
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