Contributions to catalysis and potential interactions of the three catalytic domains in a contiguous...

Contributions to catalysis and potential interactionsof the three catalytic domains in a contiguous trimericcreatine kinaseGregg G. Hoffman1, Omar Davulcu2, Sona Sona1 and 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: [email protected]

(Received 23 July 2007, revised 26 Novem-

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; kcat, 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-

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 three-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.

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

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.

Catalysis in a contiguous trimeric creatine kinase G. G. Hoffman et al.


conserved this sequence similarity for as long as

675 million years, when Chaetopterus (a lophotrocozo-

an invertebrate) last shared a common 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

C fi S mutant and no subscript corresponds to a wild-

type domain). Expression of wild-type FlgCK and sin-

gle and double C fi 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 C fi 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 C fi 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 C fi 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 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 KS(ADP) (lM) KM(ADP) (lM) KS(PCr) (mM) KM(PCr) (mM) KS ⁄ KM

Wild-type 151 ± 49.3 100 ± 0.9 3.1 ± 0.6 2.2 ± 0.5 1.4 ± 0.4

D1SD2D3 178 ± 79.8 123 ± 28.4 2.9 ± 0.8 2.1 ± 0.5 1.4 ± 0.7

D1D2SD3 109 ± 26.7 96 ± 3.3 2.1 ± 0.4 1.9 ± 0.2 1.1 ± 0.3

D1D2D3S 163 ± 13.3 104 ± 11.5 2.5 ± 0.4 1.6 ± 0.2 1.6 ± 0.3

D1SD2D3S 138 ± 41.7 90 ± 12.5 3.0 ± 0.7 2.0 ± 0.2 1.5 ± 0.2

D1D2SD3S 137 ± 33.5 112 ± 15.8 1.6 ± 0.2a 1.4 ± 0.4 1.2 ± 0.4

D1SD2SD3 173 ± 9.7 123 ± 23.2 2.5 ± 0.1 1.7 ± 0.3 1.4 ± 0.1

D1SD2SD3S 54 ± 4.5a 52 ± 4.1a 3.0 ± 0.4 2.9 ± 0.3 1.0 ± 0.1

a Values that are significantly different from wild-type (P < 0.05).



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 D1SD2D3, D1D2SD3 and D1D2D3Sproduced Vmax reductions of approximately 18, 45 and

40%, respectively (Table 2 and Fig. 2). The Vmax and

kcat values 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 D1D2SD3S and 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 D1SD2D3Smutant were significantly higher than the values for

the D1D2SD3S and 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

kcat 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

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 kcatdue 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 kcatvalues: 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). kcat values are reported for the trimeric molecule.

Construct

Vmax (lmolÆmin)1Æ

mgÆprotein)1) kcat (s)1)

kcat ⁄ KM(PCr)

(s)1ÆmM)1)

Wild-type 328 ± 16.0 715 ± 34.9 330

D1SD2D3 270 ± 9.1ab 586 ± 19.8ab 280

D1D2SD3 180 ± 5.1a 392 ± 11.1a 210

D1D2D3S 196 ± 14.0a 427 ± 30.4a 270

D1SD2D3S 75 ± 1.1ab 164 ± 2.4ab 80

D1D2SD3S 125 ± 7.6a 273 ± 16.5a 200

D1SD2SD3 113 ± 11.6a 243 ± 24.9a 140

D1SD2SD3S 0.7 ± 0.1a 1.6 ± 0.2a 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.



compared with D1D2SD3S and D1SD2SD3, and the

similar kcat values seen in D1D2SD3S and 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 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

FlgCK was expressed according to the protocol used for

other invertebrate CKs [32,33]. Bacteria were harvested by

centrifugation at 4 �C 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

N2 gas. Cellular debris was pelleted by centrifugation at

23 000 g for 20 min at 4 �C. 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 4 �Cfor 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



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

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 37 �C 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

D1SD2SD3S mutant 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 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]:

m¼Vmax½PCr�½ADP�

aKSðPCrÞKSðADPÞ þaKSðPCrÞ½ADP�þaKSðADPÞ½PCr�þ ½PCr�½ADP�

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). Vmax is 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.

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¢- to 3¢)

PCR primers

D1 forward GAG CAC AAC AAT TGG ATG GCCD1 reverse CCT TTC TCG AGT CTC TTC TCCD2 forward GAG ACT CGA GAA AGG AGA GGD2 reverse GGC AGA CGT CAG CAG TGGD3 forward CCA CTG CTG ACG TCT GCCD3 reverse ATC AGC CTA GGC CCT TTC GTC

QuikChange mutagenic primers

D1 forward CAT CCA CAC GTC CCC CAG TAA CTT AGGD1 reverse CCT AAG TTA CTG GGG GAC GTG TGG ATGD2 forward CGT GCT GAC ATC CCC CAG CAA CCT GGGD2 reverse CCC AGG TTG CTG GGG GAT GTC AGC ACGD3 forward CAT CCT GAC CTC CCC TAG CAA CCT GGGD3 reverse CCC AGG TTG CTA GGG GAG GTC AGG ATG



isoenzymes in tissues with high and fluctuating energy

demands: the ‘phosphocreatine circuit’ for cellular

energy homeostasis. Biochem J 281, 21–40.

3 Grossman SH & Sellers DS (1998) Subunit conforma-

tion and dynamics in a heterodimeric protein: studies of

the hybrid isozyme of creatine kinase. Biochim Biophys

Acta 1387, 447–453.

4 Hornemann T, Rutishauser D & Wallimann T (2000)

Why is creatine kinase a dimer? Evidence for cooper-

ativity between the two subunits. Biochim Biophys Acta

1480, 365–373.

5 Lahiri SD, Wang PF, Babbitt PC, McLeish MJ,

Kenyon GL & Allen KN (2002) The 2.1 A structure of

Torpedo californica creatine kinase complexed with the

ADP-Mg2+-NO3)-creatine transition-state analogue

complex. Biochemistry 41, 13861–13867.

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-Mg2+-NO3)-creatine transition

state analogue complex as detected by hydrogen ⁄ deute-rium exchange. Biochemistry 42, 13596–13604.

13 Forstner M, Muller A, Rognan D, Kriechbaum M &

Wallimann T (1998) Mutation of cis-proline 207 in

mitochondrial creatine kinase to alanine leads to

increased acid stability. Protein Eng 11, 563–568.

14 Uda K & Suzuki T (2004) Role of amino acid residues

on the GS region of Stichopus arginine kinase and

Danio creatine kinase. Protein J 23, 53–64.

15 Rao JK, Bujacz G & Wlodawer A (1998) Crystal struc-

ture of rabbit muscle creatine kinase. FEBS Lett 439,

133–137.

16 Furter R, Furter-Graves EM & Wallimann T (1993)

Creatine kinase: the reactive cysteine is required for

synergism but is nonessential for catalysis. Biochemistry

32, 7022–7029.

17 Reddy S, Jones AD, Cross CE, Wong PS & Van Der

Vliet A (2000) Inactivation of creatine kinase by

S-glutathionylation of the active-site cysteine residue.

Biochem J 347, 821–827.

18 Tanaka N, Tonai T & Kunugi S (1997) Site-specific

modification of rabbit muscle creatine kinase with sulf-

hydryl-specific fluorescence probe by use of hydrostatic

pressure. Biochim Biophys Acta 1339, 226–232.

19 Compaan DM & Ellington WR (2003) Functional con-

sequences of a gene duplication and fusion event in an

arginine kinase. J Exp Biol 206, 1545–1556.

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.

27 Wang PF, McLeish MJ, Kneen MM, Lee G & Kenyon

GL (2001) An unusually low pK(a) for Cys282 in the

active site of human muscle creatine kinase. Biochemis-

try 40, 11698–11705.

28 Tombes RM & Shapiro MM (1987) Enzyme termini of

a phosphocreatine shuttle. J Biol Chem 262, 16011–

16019.

29 Hawkins RJ & McLeish TC (2006) Coupling of global

and local vibrational modes in dynamic allostery of

proteins. Biophys J 91, 2055–2062.



30 Gunasekaran K, Ma B & Nussinov R (2004) Is allo-

stery an intrinsic property of all dynamic proteins?

Proteins 57, 433–443.

31 Borson ND, Salo WL & Drewes LR (1992) A lock-

docking oligo(dT) primer for 5’ and 3’ RACE PCR.

PCR Methods Appl 2, 144–148.

32 Hoffman GG, Sona S, Bertin M & Ellington WR (2006)

The role of an absolutely conserved tryptophan residue

in octamer formation and stability in mitochondrial crea-

tine kinases. Biochim Biophys Acta 1764, 1512–1517.

33 Sona S, Suzuki T & Ellington WR (2004) Cloning and

expression of mitochondrial and protoflagellar creatine

kinases from a marine sponge: implications for the ori-

gin of intracellular energy transport systems. Biochem

Biophys Res Commun 317, 1207–1214.

34 Strong SJ & Ellington WR (1996) Expression of

horseshoe crab arginine kinase in Escherichia coli and

site-directed mutations of the reactive cysteine peptide.

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.



Contributions to catalysis and potential interactions of the three catalytic domains in a contiguous...

Documents

Transcript of Contributions to catalysis and potential interactions of the three catalytic domains in a contiguous...