It is not known whether the active sites in each of the contiguous flagellar domains are catalytically competent, and, if so, whether they are capable of acting independently.. Abbreviati
Trang 1of the three catalytic domains in a contiguous trimeric
creatine kinase
Gregg G Hoffman1, Omar Davulcu2, Sona Sona1and W Ross Ellington1,3
1 Department of Biological Science, Florida State University, Tallahassee, FL, USA
2 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA
3 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA
Creatine kinase (CK) plays a central role in energy
homeostasis in cells that display high or variable rates
of ATP utilization, such as neurons, muscle fibers,
transport epithelia and spermatozoa [1] The
physio-logical roles of the CK reaction are greatly facilitated
by the presence of three nuclear gene families, each
targeted to and localized in specific intracellular
compartments – cytoplasmic (CyCK), mitochondrial
(MtCK) and flagellar (FlgCK) Two of these isoforms,
CyCK and MtCK, are oligomeric [2] Both have been
the subject of intensive research due to their
physiolog-ical importance and their utility as models for
under-standing bimolecular catalysis CyCKs are obligate
dimers, while most MtCKs function in an equilibrium
of dimers and octamers, with the latter predominating
under physiological conditions, at least in higher
organisms [2] This quaternary structure appears to be
required for catalysis in both the cytoplasmic and
mitochondrial isoforms, and there is compelling evi-dence indicating that the active sites do not function independently within a given oligomer [3–7] FlgCKs exist as contiguous trimers, with three catalytically complete domains, each with its respective N- and C-domains, fused into a single polypeptide [8,9] Struc-tural studies have not been conducted on FlgCKs, and the catalytic competence of individual domains and the potential interactions between domains remain unknown
Considerable effort has been focused on determining the physiological and functional importance of the quaternary structure in CyCKs and MtCKs, as oligo-merization is strongly correlated with intracellular localization in both [1,2] The potential for interaction between adjacent subunits in CKs has been the source
of much speculation, but recent X-ray crystallographic [5,6,10] and enzyme kinetics analyses of heterodimers
Keywords
contiguous trimer; cooperativity; domain
interaction; flagellar creatine kinase; kinetics
Correspondence
W R Ellington, Institute of Molecular
Biophysics, Florida State University,
Tallahassee, FL, USA
Fax: +1 850 644 0481
Tel: +1 850 644 5406
E-mail: elling@bio.fsu.edu
(Received 23 July 2007, revised 26
Novem-ber 2007, accepted 10 DecemNovem-ber 2007)
doi:10.1111/j.1742-4658.2007.06226.x
Three separate creatine kinase (CK) isoform families exist in animals Two
of these (cytoplasmic and mitochondrial) are obligate oligomers A third, flagellar, is monomeric but contains the residues for three complete CK domains It is not known whether the active sites in each of the contiguous flagellar domains are catalytically competent, and, if so, whether they are capable of acting independently Here we have utilized site-directed muta-genesis to selectively disable individual active sites and all possible combi-nations thereof Kinetic studies showed that these mutations had minimal impact on substrate binding and synergism Interestingly, the active sites were not catalytically equivalent, and were in fact interdependent, a phenomenon that has previously been reported only in the oligomeric
CK isoforms
Abbreviations
AK, arginine kinase; CK, creatine kinase; CyCK, cytoplasmic CK; FlgCK, flagellar CK; k cat , catalytic turnover; MtCK, mitochondrial CK; PCr, phosphocreatine; TSAC, transition state analog complex.
Trang 2of wild-type and inactive CK subunits [3,4,11]
convinc-ingly show that intra-oligomer interactions modulate
catalytic activity in a manner that has been described
as ‘flip-flop cooperativity’ in the case of chicken
cyto-plasmic CK [3,4,11]
Numerous approaches, including X-ray
crystallogra-phy [5,6], hydrogen⁄ deuterium exchange–mass
spec-trometry [12], small angle X-ray scattering [13] and
site-directed mutagenesis [14] have demonstrated that
CyCKs and MtCKs undergo substantial
conforma-tional changes upon transition from the open,
sub-strate-free state to the closed transition state analog
complex (TSAC) that is seen when MgADP, creatine
and nitrate are bound to CKs This transition involves
the movement of two flexible loops (residues 60–72
and 323–333 in both Torpedo and rabbit CyCKs) and
at the N-terminus, over distances up to 19 A˚ as the
molecule responds to occupancy of the active sites
[5,6] The homodimeric apo-crystal structure of rabbit
muscle CK consists of two identical conformational
states for the monomeric subunits in the dimer [15] In
contrast, the recently published crystal structure of the
TSAC of rabbit muscle CK [6] (and the TSAC
struc-ture for Torpedo [5]) is highly asymmetrical, with only
one of the monomers in the closed configuration
When superimposed, these asymmetrical monomers
reveal significant movement of five structural elements,
which may explain the difference between the apo and
closed states [6]
These clear, large-scale and widely dispersed
confor-mational changes pose unique constraints upon any
tertiary structure that functionally competent
contigu-ous trimers of FlgCK may potentially adopt This
pos-sibility raises some fundamental questions regarding
the connections between structure and catalysis in this
relatively unstudied molecule, i.e how can loop
move-ment and intra-subunit communication be
accommo-dated in a contiguous trimer, and, if there are
constraints, do they have an impact on catalysis in
other domains or do the domains function
indepen-dently across the molecule?
To address the above issues, we have cloned and
expressed a 1167 residue FlgCK from the marine
worm Chaetopterus variopedatus (referred to here as
CVFlgCK), and utilized site-directed mutagenesis of
the active-site cysteine residue(s) to selectively eliminate
catalysis in each of the individual domains and in all
possible combinations of domains Inactivation of this
cysteine has been shown to reduce catalytic turnover
(kcat) by > 99% compared with wild-type in
sev-eral CKs [16–18] Our results show that the mutations,
with a few exceptions, had no significant effect on
sub-strate binding and synergism Interestingly, while all
three CK domains were shown to be catalytically com-petent, they were not equivalent in terms of catalytic turnover rates More importantly, the relative contri-bution of any given active site depended on the cata-lytic state of the active site within the remaining domains Both CyCK and MtCK have been shown to undergo substantial conformational changes upon sub-strate binding, and it is reasonable to expect that simi-lar movements and interactions also occur in FlgCKs The catalytic non-equivalence reported here clearly indicates that this is indeed the case, and that these interactions may be representative of a suite of inter-actions and structural changes that are required for catalysis across this entire enzyme family
Results and Discussion
FlgCKs lack quaternary structure and are monomers that contain three apparently complete CK domains Recently, a number of other enzymes with multiple catalytic domains have been identified – two-domain arginine kinases [19–21], a two-domain carbonic anhy-drase [22], a domain luciferase [23] and a three-domain adenylate kinase [24] The present study provides insight into the inter-dependent functional properties of the three domains of FlgCK, and lays the groundwork for study of the relationship between these functional properties and the structural interac-tions that potentially mediate them
Analysis of the primary structures of the three FlgCK domains
Two CK TSAC crystal structures have been published (Torpedo and rabbit muscle) Both have one subunit in
a quasi-open, binary complex with MgADP and one in
a closed TSAC with MgADP, creatine and nitrate [5,6] This active-site asymmetry occurs even though the crystals for both Torpedo and rabbit muscle CK were grown under conditions that would strongly favor TSAC formation, indicating that, at least in mul-timeric CKs, only one monomer within a given dimer can form the TSAC, or that formation of this TSAC somehow stabilizes the open state of the adjoining active site or precludes binding of all components to form a TSAC Comparison of the two monomers within a given isoform reveals that two sets of confor-mational changes are potentially important for cataly-sis and inter-subunit communication; the first involves movements within the two loops that act to control access to the active site(s), and the second involves a significant structural change within the first 20 N-ter-minal residues
Trang 3Figure 1 shows a multiple sequence alignment in
which the sequences of the three contiguous domains
of FlgCK (ChaetFlgD1–3) are aligned with the
sequences of Torpedo and rabbit muscle CK
mono-mers The flexible loops, key catalytic residues and a
conserved proline that seems in the rabbit crystal
structure to act as a hinge point when the N-terminal
undergoes conformational changes upon conversion to
the TSAC are indicated (many of the N-terminal
resi-dues in the Torpedo structure were not well resolved
[5] and were excluded from the final model) The
speci-ficity loop (creatine binding pocket, residues 60–72 in
Torpedo) is nearly identical in all five CK domains, and the nucleotide binding loop (323–335 in Torpedo)
is quite similar (shown in blue in Fig 1) The key cata-lytic residues identified in Torpedo CK are conserved
in all three FlgCK domains (shown in red in Fig 1),
as is the ‘hinge’ proline (position 21 in Torpedo, show
in pink in Fig 1)
Based on the above comparisons, it appears that all three FlgCK domains have the requisite elements for catalysis and are at least capable of the same types of structural interactions described for oligomeric CK iso-forms It is important to note that these isoforms have
Fig 1 Multiple sequence alignment of the sequences for Torpedo [5] and rabbit mus-cle [6] CKs and each of the three FlgCK domains (ChaetFlgCKD1–3) Residues directly implicated in catalysis are shown in red, the flexible loops that have been shown
to undergo conformational changes upon substrate binding are shown in blue, and the N-terminal ‘hinge’ proline is shown in pink The highly conserved reactive cysteine residues that were the mutagenic target of this study are shown in green.
Trang 4conserved this sequence similarity for as long as
675 million years, when Chaetopterus (a
lophotrocozo-an invertebrate) last shared a common lophotrocozo-ancestor with
the deuterostomes [25] This suggests that these
struc-tural elements play an important functional role in this
enzyme system
Expression of wild-type and mutant FlgCKs
Seven mutant constructs were engineered using the
wild-type C variopedatus FlgCK as the platform All
mutations involved conversion of the reactive cysteine
residue within a domain (C299, C667 and C1052 in
CVFlgCK; see Fig 1), or a combination of domains,
to serine In this context, each FlgCK domain will be
referred to as D1, D2 and D3, respectively Previous
work has shown that this cysteine to serine mutation
dramatically reduces enzyme activity in the reverse
cat-alytic direction, especially at low Cl) concentrations
[4,16,26,27] The following combinations of mutated
domains were constructed: D1SD2D3, D1D2SD3,
D1D2D3S, D1D2SD3S, D1SD2 D3S, D1SD2SD3 and
D1SD2SD3S (where the subscript S corresponds to the
Cfi S mutant and no subscript corresponds to a
wild-type domain) Expression of wild-wild-type FlgCK and
sin-gle and double Cfi S FlgCK mutants yielded large
amounts of soluble, recombinant protein that was
eas-ily purified to homogeneity by low-pressure
chroma-tography As expected, expression of the triple mutant
(D1SD2SD3S) yielded CK with dramatically reduced
activity In fact, it was necessary to significantly
con-centrate the purified protein from 2 L of bacterial
culture to obtain sufficient recombinant triple
mutant CK for kinetic analyses
Kinetic analysis of wild-type and mutant flgCKs
Binary (KS) and ternary (KM) substrate-binding
con-stants for both ADP and phosphocreatine (PCr), as
well as the substrate-binding synergism (KS⁄ KM), were
determined for the wild-type and the seven Cfi S mutant constructs With only a few exceptions, muta-tion of the reactive cysteine had no significant impact
on KS or KM values for the recombinant flgCKs (Table 1) There was a significant decrease of KS(PCr)in the D1D2SD3S mutant as well as of the KS(ADP) and
KM(ADP) values for the triple mutant The wild-type and all mutant constructs demonstrated very limited substrate-binding synergism as evidenced by KS⁄ KM values slightly above unity Synergy values for the mutants were not significantly different from those of the wild-type Overall, our results show that Cfi S mutations in the FlgCK domains, individually and in combination, had little impact on substrate binding in the reverse catalytic direction This has also been observed for chicken [4] and human [27] cytoplas-mic CKs Interestingly, this is not the case for octa-meric mitochondrial CK, where an 11-fold increase in
KM(PCr)was reported for the Cfi S mutant [16] Our values for ADP binding in the wild-type CVFlgCK contigious trimer are similar to but somewhat lower than those reported for the oligomeric CK isoforms
KM(ADP) values range from 150 lm for MtCK octa-mers [2] to between 190 and 440 lm for Cy CK dimers This trend is more pronounced for PCr bind-ing Tombes and Shapiro reported a KM(PCr)value that
is twice that reported here [28]
In contrast to our substrate-binding parameters, the cysteine mutations in the domains of FlgCK were observed to have a profound impact on catalytic rates and relative efficiency (Table 2 and Fig 2) Pre-vious work has shown that inactivation of the reac-tive cysteine produces a dramatic reduction in Vmax and kcat for a variety of CKs [4,17,26,27], and our triple mutant, as expected, displayed very limited activity as evidenced by very low Vmax and kcat val-ues (Table 2 and Fig 2) If each CK domain of the FlgCK has an equal potential for catalytic rate enhancement, then it might be anticipated that Cfi S mutations in individual domains and
Table 1 Kinetic parameters for wild-type and mutant FlgCK constructs Values represent mean ± 1 SD (n = 3).
a Values that are significantly different from wild-type (P < 0.05).
Trang 5combinations of domains will produce proportionate
decreases in catalytic turnover
Our results clearly show that domains 1–3 are not
equal in their contributions to catalysis (Table 2) The
single mutants D1SD2D3, D1D2SD3 and D1D2D3S
produced Vmax reductions of approximately 18, 45 and
40%, respectively (Table 2 and Fig 2) The Vmax and
kcatvalues for the D1SD2D3 mutant were significantly
higher than the values for the D1D2SD3 and
D1D2D3S mutants (values for the latter two were not
different from each other) Of the double mutants, the
D1SD2 D3S mutation produced nearly an 80%
reduc-tion in catalytic rate, while D1D2SD3Sand D1SD2SD3
constructs were approximately 60 and 65% less active,
respectively, than the wild-type FlgCK (Table 2 and
Fig 2) The Vmax and kcat values for the D1SD2D3S
mutant were significantly higher than the values for
the D1D2SD3Sand D1SD2SD3 mutants (values for the
latter two were not different from each other) Because
of the minimal changes in substrate binding, catalytic efficiency (kcat⁄ KM) decreased in direct relation to the
kcatvalues (Table 2)
These results show that the contribution of each
CK domain to catalysis depends on which domains were inactivated by the Cfi S mutation, suggesting that interaction between sites has an impact on cata-lytic throughput This lack of catacata-lytic equivalence is reminiscent of recent work on contiguous dimeric argi-nine kinases (AKs) These AKs, consisting of two complete fused AK domains in a single polypeptide chain, are present in a number of metazoan groups [20] Bacterial expression of wild-type and truncated contiguous dimeric AKs showed that domain 1 had limited [21] or no [19] catalytic activity Interestingly, maximal activity of domain 2 was achieved only when domain 1 was functional, reinforcing, once again, the idea that catalysis at one active site is affected by the presence of neighboring active sites
The negative cooperativity previously reported for rabbit muscle CK [7] and the crystallographic data recently published by Lahiri et al [5] for Torpedo CK are consistent with a model in which the formation of the TSAC in one monomer affects the binding affini-ties of the second monomer within a dimer As signifi-cant movements are associated with formation of the TSAC, it is reasonable to speculate that closing of one active site is structurally linked to substrate binding in the second Stated another way, formation of the TSAC in one active site may act to stabilize the open state in the other, or preclude its closing [5] This ‘tug-of-war’ scenario, whereby the closing of one active site exerts pressure through a suite of atomic interactions
to inhibit the binding and closing of any other active sites that are in communication with the closed site, is simple and appealing and goes some distance towards explaining the asymmetry in both of the oligomeric TSAC structures published to date
It is likely that the above types of interaction play a role in the catalytic nonequivalence within the three-domain monomer reported here This type of interac-tion may explain the different kcat reductions seen in the double mutants With regard to the possibility that active sites influence adjacent active sites, domain 2 may be more sensitive to these interactions, as it is adjacent to two domains Because of this, the D1SD2 D3S mutation may be expected to have the lowest kcat due to potential constraints imposed upon it by both domains 1 and 3 Domains 1 and 3, on the other hand, only experience the constraints from domain 2, which explains two results seen in analysis of kcat values: the lower kcat seen in D1SD2 D3S when
Table 2 Enzyme turnover and relative efficiency for wild-type and
mutant FlgCK trimeric constructs Values represent mean ± 1 SD
(n = 3) kcatvalues are reported for the trimeric molecule.
Construct
Vmax(lmolÆmin)1Æ
mgÆprotein)1) kcat(s)1)
kcat⁄ K M(PCr)
(s)1Æm M )1)
D1SD2D3 270 ± 9.1 ab 586 ± 19.8 ab 280
D1D2 S D3 S 125 ± 7.6 a 273 ± 16.5 a 200
D1 S D2 S D3 113 ± 11.6a 243 ± 24.9a 140
D1SD2SD3S 0.7 ± 0.1 a 1.6 ± 0.2 a 0.6
a Values that are significantly different from wild-type (P < 0.05).
b Mutants that are significantly different from other mutants within
a given class (single or double mutants).
Fig 2 Impact of C fi S mutations of individual domains and
com-binations of domains on Vmax Percentage values represent the
per-centage of wild-type Vmax Vmax values are mean ± 1 SD (n = 3).
The superscript ‘a’ indicates values that are significantly different
from wild-type (P < 0.05) The superscript ‘b’ indicates mutants
that are significantly different from other mutants within a given
class (single or double mutants) The terminology for the mutants
is described in the text.
Trang 6compared with D1D2SD3S and D1SD2SD3, and the
similar kcatvalues seen in D1D2SD3Sand D1SD2SD3
Given the wealth of structural data available, it is
surprising that little evidence for a structural network
such as that described above exists for CK An
intrigu-ing alternative to the classical model of multidomain
interactions has been proposed by Hawkins and
McLeish [29] They present a model in which allostery
arises from coupling of changes in local vibrational
modes to changes in global entropy, in which
altera-tions in protein flexibility upon ligand binding at one
site affect the entropic cost of binding at neighboring
sites This idea stems from the fact that proteins exist as
dynamic ensembles of conformational states, and ligand
binding redistributes the population within the
ensem-ble, leading to altered conformations at other,
some-times distant, sites [29,30] These potentially distal sites
may also experience an increase in flexibility, which,
together with enthalpic contributions such as hydrogen
bond formation between substrate and enzyme, may
serve to partially offset the loss in entropy that
accom-panies substrate binding This increase in flexibility,
however, may also have the side effect of impeding
binding in adjacent active sites, essentially allowing only
one of a set of interacting active sites to complete a
catalytic cycle at a time Further understanding of
catalysis and the interaction of active sites in these
unique contiguous trimeric FlgCKs will depend on the
outcome of on-going studies of expressed truncated
contiguous dimers and monomers, as well as X-ray
crystallographic determination of the atomic structure
Experimental procedures
Amplification of full-length FlgCK cDNA
Chaetopterus variopedatus mRNA previously isolated by
our group [8] was used to amplify, clone and sequence the
FlgCK cDNA full-length transcript Briefly, single-stranded
cDNA was reverse-transcribed using Ready-to-Go You
Prime beads (GE Healthcare, Piscataway, NY, USA) and a
lock-docking oligo(dT) reverse primer [31] according to the
manufacturer’s instructions The full-length cDNA was
produced and PCR-amplified in a Hybaid PCR Sprint
thermocycler (Ashford, UK) using gene-specific primers
designed to amplify the full-length coding sequence from
the start to the stop codon using PfuTurbo Hotstart DNA
polymerase (Stratagene, La Jolla, CA, USA) PCR
amplifi-cation was carried out using a 1.5 min incubation at 95C,
followed by 17 cycles of 95C for 40 s, 60 C for 40 s, and
68C for 16 min A single PCR product was produced,
and this was gel-purified using a QiaQuick spin kit (Qiagen,
Valencia, CA, USA) This product was subcloned into a
puC19 TA (TOPO) cloning vector (Invitrogen, Carlsbad,
CA, USA), and plasmids from two independent clones were completely sequenced in both directions on an automated Applied Biosystems model 3100 genetic analyzer (Foster City, CA, USA)
Expression and purification of recombinant protein
The sequence-verified full-length CVFlgCK cDNA was ligated into the pETBlue1 vector system (EMD
Bioscienc-es⁄ Novagen, La Jolla, CA, USA), and used to transform BL21 Tuner(DE3)-pLacI expression hosts (Novagen) according to the manufacturer’s instructions Recombinant FlgCK was expressed according to the protocol used for other invertebrate CKs [32,33] Bacteria were harvested by centrifugation at 4C for 15 min at 17 000 g The pelleted cells were resuspended in lysis buffer (50 mm Tris, 300 mm NaCl, 5 mm EDTA, pH 7.8) using a Polytron homogenizer (Brinkman, Westbury, NY, USA), and then lysed using 100 cycles of microfluidization (Microfluidics, Newton, MA) in
N2gas Cellular debris was pelleted by centrifugation at
23 000 g for 20 min at 4C CK expression was verified using a reverse-direction (PCr fi ATP) spectrophotomet-ric assay as previously described [34] Expression of recom-binant wild-type FlgCK yielded substantial levels of soluble enzyme activity
Wild-type and mutant constructs of CVFlgCK were all easily purified from cellular lysates using two rounds of low-pressure chromatography Lysates were exhaustively dia-lyzed against DEAE running buffer (10 mm Tris, 0.5 mm EDTA, 1 mm DTT at pH 8.1), briefly centrifuged at 4C for 15 min at 23 000 g, and then applied to a 40 mL DEAE– Sepharose Fast Flow column(GE Biotech, Piscataway, NJ, USA) equilibrated with running buffer After washing, pro-teins were eluted with a 400 mL linear gradient of NaCl (from 0 to 250 mm in running buffer) Fractions showing
CK activity were pooled, exhaustively dialyzed against hydroxyapatite running buffer (5 mm potassium phosphate,
1 mm DTT at pH 7.0), and applied to an 80 mL Bio-Gel HT hydroxyapatite column (Bio-Rad Laboratories, Hercules,
CA, USA) After washing, proteins were eluted with a
400 mL linear gradient of 5–400 m potassium phosphate (pH 7.0) For each construct, active hydroxyapatite fractions were analyzed by SDS–PAGE [35] FlgCK fractions were pooled and concentrated using pressure filtration Protein content was determined using a Bio-Rad protein assay kit based on the Bradford method [36], using bovine serum albumin as the standard The resulting FlgCK preparations were essentially homogeneous
Site-directed mutagenesis
As Fig 2 clearly shows, the residues surrounding the reac-tive cysteines are highly conserved in all three FlgCK
Trang 7domains; therefore, they could not be directly mutated in
the full-length expression vector as the mutagenic primers
would not be domain-specific Thus, each of the domains
was excised using restriction enzymes, ligated into TOPO
cloning vectors and mutated The mutated construct was
then excised and re-ligated back into the original expression
vector containing the two non-mutated domains The
fol-lowing restriction enzymes were used to separate individual
domains: D1, MfeI and XhoI; D2, XhoI and AatII; D3,
AatII and AvrII PCR using Ex Taq HS polymerase
(Taka-ra USA, Santa Ana, CA, USA) was performed to fill in the
sticky ends and add adenine nucleotide overhangs before
ligating the individual domains into the TOPO vectors
using the primers listed in Table 3
Mutations were carried out using the QuikChange
muta-genesis kit (Stratagene) according to the manufacturer’s
protocol The specific primers used for the mutation(s) are
listed in Table 3 Briefly, the template plasmid was
ampli-fied using PfuUltra Hotstart DNA polymerase (Stratagene)
with a forward primer and its reverse complement, both
coding for the target mutation The original methylated
template plasmid was digested using the restriction enzyme
DpnI by incubating at 37C for 1 h The amplified plasmid
was then transformed into Escherichia coli XL1-Blue
super-competent cells (Stratagene) according to the
manufac-turer’s protocol Carbenicillin-resistant transformed cells
were plated, and plasmids were isolated from overnight
cul-tures grown from single colonies These plasmids were
iso-lated using a Qiagen QIAprep Spin Miniprep kit The
mutant inserts were verified by sequencing and manipulated
as described above The site-directed mutants were
expressed and purified to homogeneity as for the wild-type
The purity and protein content of the mutant FlgCKs were
determined as for the wild-type preparation All mutant
constructs yielded active soluble protein, although the
D1SD2SD3Smutant had minimal catalytic activity
Enzyme kinetics
Kinetic assays were run on a Cary 100 UV–visible spectro-photometer (Varian, Walnut Creek, CA, USA) using the manufacturer’s software Initial velocity values were deter-mined for the reverse reaction by varying the concentration
of one substrate versus six fixed concentrations of the sec-ond substrate and vice versa, resulting in a 6· 6 matrix Actual concentrations of both substrates were empirically determined by enzymatic standardization (for PCr) and spectrophotometric standardization (for ADP) Magnesium acetate was added to a concentration of 1 mm above the concentration of ADP to ensure full saturation of ADP
by Mg2+ Assay buffer (100 mm Na-HEPES, pH 7) was added to each 3 mL cuvette to bring the total reaction volume to 2.5 mL All assays were run at 25C and were nominally Cl)-free to maximize the inhibitory impact of the Cfi S mutation Kinetic rate measurements were fit to the following rate equation for a random order, sequential, bimolecular–bimolecular reaction mechanism using non-linear least-squares regression [37]:
m¼
Vmax½PCr½ADP
aKSðPCrÞKSðADPÞþ aKSðPCrÞ½ADP þ aKSðADPÞ½PCr þ ½PCr½ADP
Vmax, KS(PCr), KS(ADP) and a were simultaneously deter-mined KS(PCr)and KS(ADP)are the dissociation constants of phosphocreatine and ADP binary complexes, respectively
KM, the dissociation constant for the Michaelis complex with both phosphocreatine and ADP bound, was deter-mined from the relationship KM=a(KS) Vmaxis expressed
as specific activity, and kcat is calculated from Vmax using molecular mass and a conversion from minutes to seconds Errors of mean values for each parameter were determined
as the standard deviation of the triplicate set Data analyses were performed using sigmaplot (SPSS, Chicago, IL, USA)
Acknowledgments
This research was supported by National Science Foundation grants IOB-0130024 and IOB-0542236 to WRE and National Institutes of Health grant R01-GM077643 to OD We thank the staff of the DNA Sequencing and Molecular Cloning facilities for their assistance
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Table 3 Primers used for filling in and for C fi S mutation of
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Primer name Sequence (5¢- to 3¢)
PCR primers
D1 forward GAG CAC AAC AAT TGG ATG GCC
D1 reverse CCT TTC TCG AGT CTC TTC TCC
D2 forward GAG ACT CGA GAA AGG AGA GG
D2 reverse GGC AGA CGT CAG CAG TGG
D3 forward CCA CTG CTG ACG TCT GCC
D3 reverse ATC AGC CTA GGC CCT TTC GTC
QuikChange mutagenic primers
D1 forward CAT CCA CAC GTC CCC CAG TAA CTT AGG
D1 reverse CCT AAG TTA CTG GGG GAC GTG TGG ATG
D2 forward CGT GCT GAC ATC CCC CAG CAA CCT GGG
D2 reverse CCC AGG TTG CTG GGG GAT GTC AGC ACG
D3 forward CAT CCT GAC CTC CCC TAG CAA CCT GGG
D3 reverse CCC AGG TTG CTA GGG GAG GTC AGG ATG
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