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 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 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- 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 (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 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. 648 FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS 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: 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 D3 S (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 (D1 S D2 S D3 S ) 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 (K S ) and ternary (K M ) substrate-binding con- stants for both ADP and phosphocreatine (PCr), as well as the substrate-binding synergism (K S ⁄ K 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 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 K M(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. K M(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 K M(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 V max and k 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 S D3 109 ± 26.7 96 ± 3.3 2.1 ± 0.4 1.9 ± 0.2 1.1 ± 0.3 D1D2D3 S 163 ± 13.3 104 ± 11.5 2.5 ± 0.4 1.6 ± 0.2 1.6 ± 0.3 D1 S D2D3 S 138 ± 41.7 90 ± 12.5 3.0 ± 0.7 2.0 ± 0.2 1.5 ± 0.2 D1D2 S D3 S 137 ± 33.5 112 ± 15.8 1.6 ± 0.2 a 1.4 ± 0.4 1.2 ± 0.4 D1 S D2 S D3 173 ± 9.7 123 ± 23.2 2.5 ± 0.1 1.7 ± 0.3 1.4 ± 0.1 D1 S D2 S D3 S 54 ± 4.5 a 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 D3 and D1D2D3 S mutants (values for the latter two were not different from each other). Of the double mutants, the D1 S D2 D3 S mutation produced nearly an 80% reduc- tion in catalytic rate, while D1D2 S D3 S and D1 S D2 S D3 constructs were approximately 60 and 65% less active, respectively, than the wild-type FlgCK (Table 2 and Fig. 2). The V max and k cat values for the D1 S D2D3 S mutant were significantly higher than the values for 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 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 k cat 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 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 (lmolÆmin )1 Æ mgÆprotein )1 ) k cat (s )1 ) k cat ⁄ K M(PCr) (s )1 ÆmM )1 ) Wild-type 328 ± 16.0 715 ± 34.9 330 D1 S D2D3 270 ± 9.1 ab 586 ± 19.8 ab 280 D1D2 S D3 180 ± 5.1 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 S D2 S D3 113 ± 11.6 a 243 ± 24.9 a 140 D1 S D2 S D3 S 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 V max . Percentage values represent the per- centage of wild-type V max . 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. 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 N 2 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 °C 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, 1mm 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 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 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 D1 S D2 S D3 S 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 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 SðADPÞ ½PCrþ½PCr ½ADP V max , K S(PCr) , K S(ADP) and a were simultaneously deter- mined. K S(PCr) and K S(ADP) are the dissociation constants of phosphocreatine and ADP binary complexes, respectively. K M , the dissociation constant for the Michaelis complex with both phosphocreatine and ADP bound, was deter- mined from the relationship K M = a(K S ). V max is expressed as specific activity, and k cat is calculated from V max 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 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 Catalysis in a contiguous trimeric creatine kinase G. G. Hoffman et al. 652 FEBS Journal 275 (2008) 646–654 ª 2008 The Authors Journal compilation ª 2008 FEBS isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit’ for cellular energy homeostasis. 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