Contributions to catalysis and potential interactions
of the three catalytic domains in a contiguous trimeric
creatine kinase
Gregg G. Hoffman
1
, Omar Davulcu
2
, Sona Sona
1
and W. Ross Ellington
1,3
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

ber 2007, accepted 10 December 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.
646 FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS
of 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-

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
(k
cat
) 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

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.
G. G. Hoffman et al. Catalysis in a contiguous trimeric creatine kinase
FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS 647
Figure 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

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: D1
S
D2D3, D1D2
S
D3,
D1D2D3
S
, D1D2
S
D3
S
,D1
S
D2 D3
S
,D1
S
D2
S
D3 and
D1
S
D2
S

M
), were
determined for the wild-type and the seven C fi S
mutant constructs. With only a few exceptions, muta-
tion of the reactive cysteine had no significant impact
on K
S
or K
M
values for the recombinant flgCKs
(Table 1). There was a significant decrease of K
S(PCr)
in
the D1D2
S
D3
S
mutant as well as of the K
S(ADP)
and
K
M(ADP)
values for the triple mutant. The wild-type
and all mutant constructs demonstrated very limited
substrate-binding synergism as evidenced by K
S
⁄ K
M
values slightly above unity. Synergy values for the
mutants were not significantly different from those of

cat
for a variety of CKs [4,17,26,27], and our
triple mutant, as expected, displayed very limited
activity as evidenced by very low V
max
and k
cat
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 C fi S mutations in individual domains and
Table 1. Kinetic parameters for wild-type and mutant FlgCK constructs. Values represent mean ± 1 SD (n = 3).
Construct K
S(ADP)
(lM) K
M(ADP)
(lM) K
S(PCr)
(mM) K
M(PCr)
(mM) K
S
⁄ K
M
Wild-type 151 ± 49.3 100 ± 0.9 3.1 ± 0.6 2.2 ± 0.5 1.4 ± 0.4
D1
S
D2D3 178 ± 79.8 123 ± 28.4 2.9 ± 0.8 2.1 ± 0.5 1.4 ± 0.7
D1D2

52 ± 4.1
a
3.0 ± 0.4 2.9 ± 0.3 1.0 ± 0.1
a
Values that are significantly different from wild-type (P < 0.05).
G. G. Hoffman et al. Catalysis in a contiguous trimeric creatine kinase
FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS 649
combinations 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 D1
S
D2D3, D1D2
S
D3 and D1D2D3
S
produced V
max
reductions of approximately 18, 45 and
40%, respectively (Table 2 and Fig. 2). The V
max
and
k
cat
values for the D1
S
D2D3 mutant were significantly
higher than the values for the D1D2
S

the D1D2
S
D3
S
and D1
S
D2
S
D3 mutants (values for the
latter two were not different from each other). Because
of the minimal changes in substrate binding, catalytic
efficiency (k
cat
⁄ K
M
) decreased in direct relation to the
k
cat
values (Table 2).
These results show that the contribution of each
CK domain to catalysis depends on which domains
were inactivated by the C fi S mutation, suggesting
that interaction between sites has an impact on cata-
lytic throughput. This lack of catalytic 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

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 D1
S
D2
D3
S
mutation may be expected to have the lowest k
cat
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 k
cat
values: the lower k
cat
seen in D1
S
D2 D3
S
when
Table 2. Enzyme turnover and relative efficiency for wild-type and
mutant FlgCK trimeric constructs. Values represent mean ± 1 SD
(n = 3). k
cat
values are reported for the trimeric molecule.
Construct
V
max

a
392 ± 11.1
a
210
D1D2D3
S
196 ± 14.0
a
427 ± 30.4
a
270
D1
S
D2D3
S
75 ± 1.1
ab
164 ± 2.4
ab
80
D1D2
S
D3
S
125 ± 7.6
a
273 ± 16.5
a
200
D1

. V
max
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.
Catalysis in a contiguous trimeric creatine kinase G. G. Hoffman et al.
650 FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS
compared with D1D2
S
D3
S
and D1
S
D2
S
D3, and the
similar k
cat
values seen in D1D2
S
D3
S
and D1
S
D2
S
D3.

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 95 °C,
followed by 17 cycles of 95 °C for 40 s, 60 °C for 40 s, and
68 °C 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

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
G. G. Hoffman et al. Catalysis in a contiguous trimeric creatine kinase
FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS 651
domains; 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

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 Mg
2+
. 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 25 ° C and were
nominally Cl
)
-free to maximize the inhibitory impact of
the C fi 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]:
m ¼
V
max
½PCr½ADP
aK
SðPCrÞ
K
SðADPÞ
þ aK
SðPCrÞ
½ADPþaK

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.
References
1 Ellington WR (2001) Evolution and physiological roles
of phosphagen systems. Annu Rev Physiol 63, 289–325.
2 Wallimann T, Wyss M, Brdiczka D, Nicolay K &
Eppenberger HM (1992) Intracellular compartmenta-
tion, structure and function of creatine kinase
Table 3. Primers used for filling in and for C fi S mutation of indi-
vidual FlgCK domains.
Primer name Sequence (5¢-to3¢)
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

6 Ohren JF, Kundracik ML, Borders CL Jr, Edmiston P
& Viola RE (2007) Structural asymmetry and intersub-
unit communication in muscle creatine kinase. Acta
Crystallogr D Biol Crystallogr 63, 381–389.
7 Price NC & Hunter MG (1976) Non-identical behaviour
of the subunits of rabbit muscule creatine kinase. Bio-
chim Biophys Acta 445, 364–376.
8 Suzuki T, Mizuta C, Uda K, Ishida K, Mizuta K, Sona
S, Compaan DM & Ellington WR (2004) Evolution
and divergence of the genes for cytoplasmic, mito-
chondrial, and flagellar creatine kinases. J Mol Evol 59,
218–226.
9 Wothe DD, Charbonneau H & Shapiro BM (1990) The
phosphocreatine shuttle of sea urchin sperm: flagellar
creatine kinase resulted from a gene triplication. Proc
Natl Acad Sci USA 87, 5203–5207.
10 Eder M, Schlattner U, Becker A, Wallimann T, Kabsch
W & Fritz-Wolf K (1999) Crystal structure of brain-
type creatine kinase at 1.41 A
˚
resolution. Protein Sci 8,
2258–2269.
11 Lin L, Perryman MB, Friedman D, Roberts R & Ma
TS (1994) Determination of the catalytic site of creatine
kinase by site-directed mutagenesis. Biochim Biophys
Acta 1206, 97–104.
12 Mazon H, Marcillat O, Forest E & Vial C (2003)
Changes in MM-CK conformational mobility upon for-
mation of the ADP-Mg
2+

20 Suzuki T, Kawasaki Y, Unemi Y, Nishimura Y, Soga
T, Kamidochi M, Yazawa Y & Furukohri T (1998)
Gene duplication and fusion have occurred frequently
in the evolution of phosphagen kinases – a two-domain
arginine kinase from the clam Pseudocardium sachalin-
ensis. Biochim Biophys Acta 1388, 253–259.
21 Suzuki T, Tomoyuki T & Uda K (2003) Kinetic proper-
ties and structural characteristics of an unusual two-
domain arginine kinase of the clam Corbicula japonica.
FEBS Lett 533, 95–98.
22 Leggat W, Dixon R, Saleh S & Yellowlees D (2005) A
novel carbonic anhydrase from the giant clam Tridacna
gigas contains two carbonic anhydrase domains. FEBS
J 272, 3297–3305.
23 Liu L, Wilson T & Hastings JW (2004) Molecular evo-
lution of dinoflagellate luciferases, enzymes with three
catalytic domains in a single polypeptide. Proc Natl
Acad Sci USA 101, 16555–16560.
24 Kinukawa M, Nomura M & Vacquier VD (2007) A sea
urchin sperm flagellar adenylate kinase with triplicated
catalytic domains. J Biol Chem 282, 2947–2955.
25 Doolittle RF, Feng DF, Tsang S, Cho G & Little E
(1996) Determining divergence times of the major king-
doms of living organisms with a protein clock. Science
271, 470–477.
26 Buechter DD, Medzihradszky KF, Burlingame AL &
Kenyon GL (1992) The active site of creatine kinase.
Affinity labeling of cysteine 282 with N-(2,3-epoxy-
propyl)-N-amidinoglycine. J Biol Chem 267, 2173–
2178.

Comp Biochem Physiol B Biochem Mol Biol 113,
809–816.
35 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
36 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein–dye binding. Anal
Biochem 72, 248–254.
37 Segel IH (1975) Enzyme Kinetics – Behavior and
Analysis of Rapid Equilibrium and Steady-State Enzyme
Systems. Wiley, New York, NY.
Catalysis in a contiguous trimeric creatine kinase G. G. Hoffman et al.
654 FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS