Dimeric Arginine Kinase Research Articles

Testing the versatility of the alternating sites of reactivity mechanism in the phosphagen kinases (768.11)

Phosphagen kinases achieve “buffering” of cellular ATP levels by catalyzing the reversible transfer of a phosphoryl group from ATP to a guanidino acceptor to form a high energy compound, phosphagen. Creatine kinase (CK), a PK found mainly in vertebrates, exists as a dimer in muscles; whereas, arginine kinase (AK), found exclusively in invertebrates, exists mainly as a monomer. The physiological functions of different oligomerization states within the PK family are not fully understood. Crystal structures of dimeric CK and AK are consistent with cooperativity in ligand binding between the two subunits. Based on these results, a reciprocating mechanism of catalysis was suggested for dimeric PKs. However, the exact mechanism and the potential effects of negative cooperativity on ligand binding ability of dimeric PKs remains unexplained. In this study, we’ve shown direct functional evidence of negative cooperativity in rabbit muscle CK (rbCK) by measuring the enzymes’ affinity for MgADP using isothermal titration calorimetry. Measurements of rbCK‐ligand interactions, in presence of saturating concentrations of creatine and nitrate, to form the transition‐state analogue quaternary complex, were fitted using a two‐site binding model with Kd1 = 56 ± 32 µM and Kd2 = 2.7 ± 1.4 mM. In the absence of creatine and nitrate, MgADP binding by rbCK fits a one‐site binding model, with Kd = 720 ± 330 µM. These suggest that the presence of creatine enables the conformation necessary to permit cross‐talk between the subunits. Pre‐steady state kinetic assays and the effect of viscosity on kcat show that the rate‐limiting step of reaction catalyzed by PKs is dependent on the oligomerization state of the enzyme, consistent with the alternating site of reactivity mechanism.Grant Funding Source: NSF Grant #0619123

The D14 and R138 ion pair is involved in dimeric arginine kinase activity, structural stability and folding

Arginine kinase (AK) is a key enzyme in cellular energy metabolism of invertebrates. There are two conserved amino acid residues D14 and R138 in dimeric AK which form inter-subunit hydrogen bond. In Stichopus japonicus AK, mutations in these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Mutations (R138G, R138A and D14G) abolished D14 and R138 interaction disrupted the structure or conformation of S. japonicus AK. These R138G, R138A and D14G mutations changed their native assembles of dimeric AK and caused them in a partially unfolded state. The partially unfolded state of these mutant AKs made them prone to aggregate under environmental stress. The D14E/R138K and R138K mutant AKs showed similar characteristics to those of WT AK for forming the interaction which could replacement roles of D14 and R138 interaction. These results suggested that D14 and R138 interaction is involved in AK's activity, substrate synergism and structural stability.

Impact of intra-subunit interactions on the dimeric arginine kinase activity and structural stability

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. In this research, six conserved residues located on the intra-subunit domain–domain interfaces were mutated to explore their roles in the activity and structural stability of dimer AK. The mutations D69A, E70A, E71A and F80A led to pronounced loss of AK activity and structural stability. Although the mutations V75A and F76A had little effect on AK activity and structure, they caused gradually decreased the stability and reactivation of dimer AK. Our results suggested that the mutations might affect the correct positioning of the N-loop and C-loop thus disrupted the efficient recognition and interactions between the N-terminal domain and C-terminal domain which may influence the compact dimer structure, and result in decreased activity and structural stability.

Impact of inter-subunit interactions on the dimeric arginine kinase activity and structural stability

Arginine kinase (AK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphoarginine to ADP in invertebrates. In this study, the inter-subunit hydrogen bonds between the Q53 and D200 and between D57 and D200 were disrupted to explore their roles in the activity and structural stability of Stichopus japonicus ( S. japonicus) AK. Mutating Q53 and/or D57 to alanine (A) can cause pronounced loss of activity and substrate synergism, and cause distinct conformational changes. Spectroscopic experiments indicated that mutations destroying the inter-subunit hydrogen bonds impaired the structure of dimer AK, and resulted in a partially unfolded state. The inability to fold to the functional compact state made the mutants prone to be inactivated and aggregate under environmental stresses. Restoring hydrogen bonds in Q53E and D57E mutants could rescue the loss of activity and substrate synergism, and conformational changes. All those results suggested that the inter-subunit interactions played a key role in keeping the activity, substrate synergism and structural stability of dimer AK. The result herein may provide a clue in understanding the folding and self-assembly processes of oligomeric proteins.

Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate

Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn²(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn²(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn²(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn²(+) on metabolic enzymes in marine invertebrates.

Characterization of a dimeric Arginine kinase in the protozoan, Phytophthora sojae, suggests an early origin of Phosphagen kinase dimers

The Phosphagen kinase (PK) enzyme family plays a critical role in energy homeostasis by catalyzing the reversible transfer of a high‐energy phosphoryl group from ATP to a guanadino‐containing acceptor molecule. Creatine kinase (CK), found in vertebrates and some invertebrates, exists as a dimer. Arginine kinase (AK), found in invertebrates, protozoa, and some bacteria, usually exists as a monomer, but several dimeric AKs have been characterized. The evolutionary relationship between these two forms of AK and the CK is unclear. Dimeric AKs described previously in invertebrates were proposed to have derived from CK, however multiple separate occurrences of this ‘back evolution’ make this scenario problematic. Using sequence motifs known through previous work to be important for dimerization in PKs, we identified an AK in a protozoan, the oomycete Phytophthora sojae, that we predicted would be dimeric. This putative dimeric AK has been cloned and expressed and shown to exhibit AK activity. More importantly, preliminary size‐exclusion chromatography data indicate that it is likely a dimeric enzyme. To expand this characterization, isothermal titration calorimetry will be used to determine whether the enzyme exhibits subunit cooperativity. These data imply that dimeric AKs were present much earlier than previously thought and suggest a different evolutionary history for PK enzymes than previously proposed.

Creatine kinases: a cornerstone for structural research in the phosphagen kinase family

In their recently published manuscript, Wu et al. (1) solved a 1.75 A structure of a dimeric arginine kinase (AK) and propose a mechanism for negative cooperativity in dimeric phosphagen kinases (PK) in general. The authors correctly cite that such AK dimers probably evolved from another PK family member, a dimeric creatine kinase (CK)-like ancestor (2), that CKs are the most studied family members for their multiple implications in human pathologies, and that their results have repercussions for the understanding of CK function. Unfortunately, some fundamental results concerning the structure/function relationship of CKs have been omitted. Pertinent to this work is the historically first X-ray structure of a PK proper, that is the large cuboidal octamer of sarcomeric mitochondrial CK with bound ADP (3), whose coordinates have been used to solve many subsequent PK structures by molecular replacement, as well as the first X-ray structures of two other CK isoforms. These include octameric ubiquitous mitochondrial CK (4) and dimeric human brain BB-CK (5), solved at 1.41 A, the highest resolution obtained so far within the PK family. These structures allowed already 13 years ago to conclude on separated active sites within the CK homooligomers and to examine the monomer/monomer interface. Even before, in 1994, the importance of the N-termini in CK protomer communication has been shown by mutational analysis (6), similar to what is presented by Wu et al. (1). Cooperativity between CK monomers within a functional CK dimer was demonstrated (7) and the conformational change in CK upon substrate (TSAC) binding from an open to a closed conformation elaborated by in-solution X-ray scattering (8). Recent reviews on these results are available (9–11). REFERENCES

Structural basis for a reciprocating mechanism of negative cooperativity in dimeric phosphagen kinase activity

Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 A, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widely than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase.

Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi—Evidence of two active domains

The cDNA and deduced amino acid sequences for arginine kinase (AK) from the deep-sea clam Calyptogena kaikoi have been determined revealing an unusual two-domain (2D) structure with molecular mass of 80 kDa, twice that of normal AK. The amino acid sequences of both domains contain most of the residues thought to be required for substrate binding found in the horseshoe crab Limulus polyphemus AK, a well studied system for which several X-ray crystal structures exist. However, two highly conserved residues, D62 and R193, that form a salt bridge thereby stabilizing the substrate-bound structure have been replaced by G and N in domain 1, and G and P in domain 2, respectively. The present effort probes whether both domains of Calyptogena AK are catalytically competent. Recombinant constructs of the wild-type enzyme of both single domains, and of selected mutants of the Calyptogena AK have been expressed as fusion proteins with the maltose-binding protein. The wild-type two-domain enzyme (2D[WT]) had high AK activity ( k cat = 23 s − 1, average value of the two domains), and the single domain 2 (D2[WT]) showed 1.5-times higher activity ( k cat = 38 s − 1) than the wild-type 2D[WT]. Interestingly, the single domain 1 (D1[WT]) showed only a very low activity ( k cat ∼ 0.016 s − 1). Introduction of a Y68A mutation in both domains virtually abolished catalytic activity. On the other hand, significant residual activity was observed ( k cat = 2.8 s − 1), when the Y68A mutation was introduced only into domain 2 of the two-domain enzyme. A similar mutation in domain 1 of the two-domain enzyme reduced activity to a much lower extent ( k cat = 11.1 s − 1). Although the domains of this “contiguous” dimeric AK each have catalytic capabilities, the presence of domain 2 strongly influences the stability and activity of domain 1.

Directed Evolution of Arginine Kinase Oligomerization

A new technique for directed evolution of protein oligomerization was developed. The system is based on fusing a target protein to the DNA binding domain of a cI lambda phage inhibitor protein lacking an oligomerization domain required for function (Mariño‐Ramírez, et al. J. Bact. 186:1311). Activation of the λ inhibitor can only proceed if the target undergoes mutational events that lead to dimerization, thus providing a selectable means for protein oligomerization. The phosphogen kinase (PK) family was used as a model system to test the technique. Within the PK family, arginine kinase (AK) is a monomer, yet the other prominent member, creatine kinase, is a homodimer. The two share a high degree of tertiary structure similarity making them a target for directed evolution to create a dimer from monomeric AK. Prior to random mutagenesis and screening, a cloned variant of L. polyphemus AK that possessed ten site‐directed mutations was created to incorporate key residues found within the dimer interface of CK, however these mutations alone do not produce a dimeric AK. Ethylenenitrosourea was used to randomly mutate the AK gene in vivo. After mutation steps, cells containing the fusion were exposed to λKH5‐h80 with increasing levels of stringency by adding variable levels of ITPG as a second means of inhibitor activation. The AKs of surviving clones were sequenced to determine the number and identity of the mutational events.

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I 1 (existing in 1.8–1.4 M urea) and I 2 (existing in 0.8–0.4 M urea). I 1 was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I 2 was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates ( I B). Comparison of the properties of these intermediates suggested that I B in the kinetic process corresponded to I 1 in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.

Two fused proteins combining Stichopus japonicus arginine kinase and rabbit muscle creatine kinase

Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold K(m) values for arginine of native AK, as well as about 50% CK activity and about 2-fold K(m) values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK.

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu 314 decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe 315 mutants including the AAloop which retained Glu 314 had modest AK activity (5–84% of the wild type). All the results above suggested that Glu 314 played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu 314 mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe 315 in dimeric AK has a different role from His 315 in monomeric AK. This might contribute to the stabilization of the native conformation, while His 315 in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.

A mechanistic role for protein oligomerization in the phosphagen kinase family

Creatine and arginine kinases catalyze reversible phosphoryl transfer between ATP and creatine or arginine, respectively. Although these two kinases share approximately 80% sequence identity, the quaternary structure of arginine kinase (AK) is typically monomeric, whereas that of creatine kinase (CK) is dimeric or octomeric. Previous structural and functional studies of dimeric CK show that the binding of a transition state analogue complex [Cr-NO3-MgADP] by the two active sites displays negative cooperativity. The goal of this project is to determine whether protein oligomerization, specifically negative cooperativity, plays a functional role in the mechanism of these enzymes. Viscosity (trehalose) variation studies and pre-steady state kinetic analyses by rapid quench of the reactions catalyzed by wild-type dimeric CK, the R147A/R151A monomeric CK and an unusual dimeric AK from Stichopus japonicus, were conducted to examine the rate-determining step of each reaction. Collectively, the results show that the rate of the reaction catalyzed by the monomeric enzymes is limited by the rate of product release, whereas that of the dimeric enzymes is limited by phosphoryl transfer. Thus, negative cooperativity within the phosphagen kinase family enhances catalytic efficiency by decreasing the free energy barrier for product release. This work was supported by NSF grant #0344432.

Evolution of the arginine kinase gene family

Arginine kinase (AK), catalyzing the reversible transfer of phosphate from MgATP to arginine yielding phosphoarginine and MgADP, is widely distributed throughout the invertebrates and is also present in certain protozoa. Typically, these proteins are found as monomers targeted to the cytoplasm, but true dimeric and contiguous dimeric AKs as well as mitochondrial AK activities have been observed. In the present study, we have obtained the sequences of the genes for AKs from two distantly related molluscs—the cephalopod Nautilus pompilius and the bivalve Crassostrea gigas. These new data were combined with available gene structure data (exon/intron organization) extracted from EST and genome sequencing project databases. These data, comprised of 23 sequences and gene structures from Protozoa, Cnidaria, Platyhelminthes, Mollusca, Arthropoda and Nematoda, provide great insight into the evolution and divergence of the AK family. Sequence and phylogenetic analyses clearly show that the AKs are homologous having arisen from some common ancestor. However, AK gene organization is highly divergent and variable. Molluscan AK genes typically have a highly conserved six-exon/five-intron organization, a structure that is very similar to that of the platyhelminth Schistosoma mansoni Arthropod and nematode AK genes have fewer introns, while the cnidarian and protozoan genes each display unique exon/intron organization when compared to the other AK genes. The non-conservative nature of the AK genes is in sharp contrast to the relatively high degree of conservation of intron positions seen in a homologous enzyme creatine kinase (CK). The present results also show that gene duplication and subsequent fusion events forming unusual two-domain AKs occurred independently at least four times as these contiguous dimers are present in Protozoa, Cnidaria, Platyhelminthes and Mollusca. Detailed analyses of the amino acid sequences indicate that two AKs (one each from Drosophila and Caenorhabditis) have what appear to be N-terminal mitochondrial targeting sequences, providing the first evidence for true mitochondrial AK genes. The AK gene family is ancient and the lineage has undergone considerable divergence as well as multiple duplication and fusion events.

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.

Thermal Inactivation and Unfolding of a Dimeric Arginine Kinase

Thermal inactivation and unfolding of the dimeric arginine kinase (AK) from sea cucumber Stichopus japonicus was investigated. The activation energy was calculated to be 388 kJ/mol. Based on the analysis of the denaturation course at 58 degrees C, a model is suggested for the thermal unfolding of this dimeric AK. In addition, the effect of free Mg(2+) and the potential biological significance on the thermal unfolding of dimeric AK is discussed.

Intermediates in the inactivation and unfolding of dimeric arginine kinase induced by GdnHCl.

Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.

The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability

Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein folding.

Arginine kinase evolved twice: evidence that echinoderm arginine kinase originated from creatine kinase

Arginine kinase (AK) was isolated from the longitudinal muscle of the sea cucumber Stichopus japonicus. Unlike the monomeric 40 kDa AKs from molluscs and arthropods, but like the cytoplasmic isoenzymes of vertebrate creatine kinase (CK), the Stichopus enzyme was dimeric. To explore the evolutionary origin of the dimeric AK, we determined its cDNA-derived amino acid sequence of 370 residues. A comparison of the sequence with those of other enzymes belonging to the phosphagen kinase family indicated that the entire amino acid sequence of Stichopus AK is apparently much more similar to vertebrate CKs than to all other AKs. A phylogenetic tree also strongly suggests that the Stichopus AK has evolved from CK. These results support the conclusion that AK evolved at least twice during the evolution of phosphagen kinases: first at an early stage of phosphagen kinase evolution (its descendants are molluscan and arthropod AKs) and secondly from CK later in metazoan evolution. A comparison of the amino acid sequence around the guanidino specificity (GS) region (which is a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) of the Stichopus enzyme with those of other phosphagen kinases showed that the GS region of the Stichopus enzyme was of the AK type: five amino acid deletions in the flexible loop region that might help to accommodate larger guanidine substrates in the active site. The presence of the AK-type deletions in the Stichopus AK, even though it seems that the enzyme's most immediate ancestor was probably CK, strongly suggests that the GS region has a role in substrate specificity. Stichopus AK and presumably other echinoderm AKs seem to have evolved from the CK gene; the sequence of GS region might have been replaced by the AK type via exon shuffling. The presence of an intron near the GS region in the Stichopus AK gene supports this hypothesis.