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 Glu³¹⁴ decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe³¹⁵ mutants including the AAloop which retained Glu³¹⁴ had modest AK activity (5–84% of the wild type). All the results above suggested that Glu³¹⁴ 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 Glu³¹⁴ 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 Phe³¹⁵ in dimeric AK has a different role from His³¹⁵ in monomeric AK. This might contribute to the stabilization of the native conformation, while His³¹⁵ 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.     

Hypotaurocyamine kinase evolved from a gene for arginine kinase

Hypotaurocyamine kinase (HTK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase (AK). HTK is found only in sipunculid worms, and it shows activities for both the substrates hypotaurocyamine and taurocyamine. Determining how HTK evolved in sipunculids is particularly insightful because all sipunculid-allied animals have AK and only some sipunculids have HTK. We determined the cDNA sequence of HTK from the sipunculid worm Siphonosoma cumanense for the first time, cloned it in pMAL plasmid and expressed it in E. coli as a fusion protein with maltose-binding protein. The cDNAderived amino acid sequence of Siphonosoma HTK showed high amino acid identity with molluscan AKs. Nevertheless, the recombinant enzyme of Siphonosoma HTK showed no activity for the substrate arginine, but showed activity for taurocyamine. Comparison of the amino acid sequences of HTK and AK indicated that the amino acid residues necessary for the binding of the substrate arginine in AK have been completely lost in Siphonosoma HTK sequence. The phylogenetic analysis indicated that the HTK amino acid sequence was placed just outside the molluscan AK cluster, which formed a sister group with the arthropod and nematode AKs. These results suggest that Siphonosoma HTK evolved from a gene for molluscan AK. Moreover, to confirm this assertion, we determined by PCR that the gene for Siphonosoma HTK has a 5-exon/4-intron structure, which is homologous with that of the molluscan AK genes. Further, the positions of splice junctions were conserved exactly between the two genes. Thus, we conclude that Siphonosoma HTK has evolved from a primordial gene for molluscan AK.     

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.     

Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase

Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of the linker in the folding, stability and function of multidomain proteins. In this research, muscle creatine kinase (CK), a dimeric two-domain protein, was used as a model protein to investigate the role of the linker in CK activity, stability and folding by mutational analysis. Two of the three mutations, L115D and L121D, resulted in a gradual decrease in CK activity and secondary structures, but did not affect CK inactivation induced by heat or guanidine hydrochloride (GdnHCl). The mutations also caused much more serious aggregation during heat- and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, none of the three mutants could successfully recover their activities by dilution-initiated refolding, and the rate constant of CK refolding was gradually decreased by the mutations. These results suggested that mutations of the hydrophobic residues in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the two domains. The results herein indicated that in addition to its role in the in vivo functions, the linker also played a crucial role in the stability and folding of CK.     

Mechanism of inhibition of arginine kinase by flavonoids consistent with thermodynamics of docking simulation

Arginine kinase plays a vital role in invertebrate homeostasis by buffering ATP concentrations. Arginine kinase might serve as a target for environmentally friendly insect-selective pesticides, because it differs notably from its counterpart in vertebrates, creatine kinase. In this study, two members of the flavonoid family, quercetin (QU) and luteolin (LU), were identified as novel noncompetitive inhibitors of locust arginine kinase. They were found to have inhibition parameters (K_i) of 11.2 and 23.9 μM, respectively. By comparing changes in the activity and intrinsic fluorescence of AK, the inhibition mechanisms of these flavonoids were found to involve binding to Trp residues in the active site. This was determined by examination of the static quenching parameter K_sv. The main binding forces between flavonoids and AK were found to be hydrophobic based on the thermodynamic parameters of changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG) and on docking simulation results. Molecular docking analyses also suggested that flavonoids could bind to the active site of AK and were close to the Trp 221 in active site. Molecular simulation results mimic the experimental results, indicated that QU had a lower binding energy and a stronger inhibitory effect on AK than LU, suggesting that the extra hydroxyl group in QU might increase binding ability.     

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.     

Creatine kinase and mitochondrial respiration in hearts of trout,cod and freshwater turtle

The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent K_m for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the K_m. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent K_m for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this K_m in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on K_m in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.Abbreviations ACR acceptor control ratio - CK creatine kinase - PCr creatine phosphate - V_ADP ADP-stimulated respiration rate - V_max maximal respiration rate - V₀ respiration rate in the absence of ADPCommunicated by: G. Heidmaier     

Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide

Arginine kinase (AK) from the horseshoe crab Limulus polyphemus was expressed in Escherichia coli. The bulk of expressed protein resided in insoluble inclusion bodies. However, approximately 3 mg enzyme protein/l culture was present as active soluble AK. The AK-containing expression vector construct was subjected to site-directed mutagenesis via a polymerase chain reaction-based megaprimer protocol. The AK reactive cysteine peptide was engineered so that it was identical to the corresponding peptide sequence of creatine kinase, another member of the guanidino kinase enzyme family. The resulting expressed protein had a considerably reduced specific activity but was still specific for arginine/arginine phosphate. No catalytic activity was observed with other guanidine substrates (creatine, glycocyamine, taurocyamine, lombricine). The reactive cysteine peptide, characteristic of all guanidino kinases, very likely plays a minimal role in determining guanidine specificity.     

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.     

Crystal structure of brain-type creatine kinase at 1.41 A resolution

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.     

Adenylate kinase: Kinetic behavior in intact cells indicates it is integral to multiple cellular processes

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by ¹⁸O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by ¹⁸O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K⁺ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2) ADP.     

The role of detergent in refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus Chinensis: the solubilization of aggregate and refolding in detergent solutions.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.     

Despite its high similarity with monomeric arginine kinase, muscle creatine kinase is only enzymatically active as a dimer

Although having highly similar primary to tertiary structures, the different guanidino kinases exhibit distinct quaternary structures: monomer, dimer or octamer. However, no evidence for communication between subunits has yet been provided, and reasons for these different levels of quaternary complexity that can be observed from invertebrate to mammalian guanidino kinases remain elusive. Muscle creatine kinase is a dimer and disruption of the interface between subunits has been shown to give rise to destabilized monomers with slight residual activity; this low activity could, however, be due to a fraction of protein molecules present as dimer. CK monomer/monomer interface involves electrostatic interactions and increasing salt concentrations unfold and inactivate this enzyme. NaCl and guanidine hydrochloride show a synergistic unfolding effect and, whatever the respective concentrations of these compounds, inactivation is associated with a dissociation of the dimer. Using an interface mutant (W210Y), protein concentration dependence of the NaCl-induced unfolding profile indicates that the active dimer is in equilibrium with an inactive monomeric state. Although highly similar to muscle CK, horse shoe crab (Limulus polyphemus) arginine kinase (AK) is enzymatically active as a monomer. Indeed, high ionic strengths that can monomerize and inactivate CK, have no effect on AK enzymatic activity or on its structure as judged from intrinsic fluorescence data. Our results indicate that expression of muscle creatine kinase catalytic activity is dependent on its dimeric state which is required for a proper stabilization of the monomers.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k_i), increasing the transition free energy changes (ΔΔG_i) and increasing the value for the midpoint of denaturation (C_m). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.     

Cytoplasmic and Mitochondrial Creatine Kinases from the Skeletal Muscle of Sperm Whale (Physeter macrocephalus). Molecular Cloning and Enzyme Characterization

We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of sperm whale MCK and sMiCK showed 94–96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods (neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K _m, K _d and k _cat) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that sperm whale MCK has a comparable affinity for creatine (K _m^Cr = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the MCK and sMiCK of sperm whale display a synergistic substrate binding (K _d /K _m = 3.1–7.8) like those of other mammalian CKs.     

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L^− 1 of LB medium. Some parameters for the renatured and soluble forms of AK, including K_m, K_d, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest k_cat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.     

Gene structure of two-domain arginine kinases from Anthopleura japonicus and Pseudocardium sachalinensis

Unusual two-domain arginine kinases (AKs) arose independently at least two times during molecular evolution of phosphagen kinases: AKs from the primitive sea anemone Anthopleura japonicus and from the clam Pseudocardium sachalinensis. To elucidate its unusual evolution, the structures of Anthopleura and Pseudocardium AK genes have been determined. The Anthopleura gene consisted of 4 exons and 3 introns: two domains are linked by a bridge intron, and each domain contains one intron in different positions. On the other hand, the Pseudocardium gene consisted of 10 exons and 9 introns: two domains are also linked by a bridge intron, and domains 1 and 2 contains 3 and 5 introns, respectively, of which 3 introns are located in exactly same positions. Since the two domains of Pseudocardium AK are estimated to have diverged about 290 million years ago, the 3 introns have been conserved at least for this long. Comparison of intron positions in Anthopleura, Pseudocardium and C. elegans AK genes indicates that there is no intron conserved through the three AK lineages, in sharp contrast to relatively conservative intron positions in creatine kinase (CK) gene family.     

The role of Cys271 in conformational changes of arginine kinase

Arginine kinase (AK), a crucial enzyme in energy metabolism, buffers cellular ATP levels by catalyzing the reversible phosphoryl transfer between ATP and arginine. To better understand the role of Cys271 in conformational changes of AK from greasyback shrimp (Metapenaeus ensis), we replaced the residue with serine and alanine. A detailed comparison of the catalytic activity and conformation was made between wild-type AK and the mutants by means of activity analysis, ultraviolet (UV) difference, fluorescence spectrum and size exclusion chromatography (SEC). The results indicated that the catalytic activity of the two mutants was gone. The substrates, arginine-ADP-Mg²⁺ could induce conformational changes, and additional NO₃⁻ could induce further changes in both the native enzyme and the variants. We speculated that Cys271 might be located in the hinge region between the two domains of AK and cause enzyme conformational changes upon addition of substrate.