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.     

Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.     

Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

The role of creatine kinase and arginine kinase in muscle.

Arginine and creatine kinase activities in different muscles are compared with calculated maximum rates of ATP turnover. The magnitude of the kinase activities decreases in the following order: anaerobic muscles and vertebrate skeletal muscles greater than heart muscle greater than insect flight muscle. The maximum activity of phosphagen kinases (i.e. creatine kinase and arginine kinase), in the direction of phosphagen formation, is lower than the calculated maximum rate of ATP turnover in insect flight muscle or rat heart.     

Arginine kinase in Phytomonas, a trypanosomatid parasite of plants

Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer.     

Variable intron/exon structure in the oligochaete lombricine kinase gene

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.     

Purification and characterization of arginine kinase from sea-urchin eggs

In most invertebrates, creatine kinase is replaced by arginine kinase, which catalyzes reversibly the transfer of a phosphate group between adenosine triphosphate and arginine. In sea-urchin larvae, arginine kinase only is expressed whereas in adult sea-urchins both arginine kinase and creatine kinase can be found in the same tissue. In order to study their developmental regulation and properties, we have purified arginine kinase to homogeneity from the eggs of the sea-urchin Paracentrotus lividus. The purification involves ethanol and ammonium sulfate precipitations, followed by an anion-exchange chromatography, an affinity chromatography and a gel filtration. A 500-fold increase in specific activity leads to a specific activity of 360 IU/mg protein at 25 degrees C. Arginine kinase (pI = 5.7) is rapidly and irreversibly inactivated at 45 degrees C. Amino acid composition and Km values (2.08 mM for phospho-L-arginine and 1.25 mM for ADP) are also given. Determination of molecular mass by gel filtration and separation by SDS/polyacrylamide gel electrophoresis indicate that the enzyme is an 81-kDa dimer of two subunits of 42 kDa.     

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.     

The structure of lombricine kinase: implications for phosphagen kinase conformational changes

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.     

Dynamical properties of the loop 320s of substrate-free and substrate-bound muscle creatine kinase by NMR: evidence for independent subunits

Muscle creatine kinase (MCK; EC2.7.3.2) is a 86 kDa homodimer that belongs to the family of guanidino kinases. MCK has been intensively studied for several decades, but it is still not known why it is a dimer because this quaternary structure does not translate into obvious structural or functional advantages over the homologous monomeric arginine kinase. In particular, it remains to be demonstrated whether MCK subunits are independent. Here, we describe NMR chemical-shift perturbation and relaxation experiments designed to study the active site 320s flexible loop of this enzyme. The analysis was performed with the enzyme in its ligand-free and MgADP-complexed forms, as well as with the transition-state analogue abortive complex (MCK-Mg-ADP-creatine-nitrate ion). Our data indicate that each subunit can bind substrates independently.     

Evolution of the diverse array of phosphagen systems present in annelids

Annelids as a group express a variety of phosphagen kinases including creatine kinase (CK), glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. In prior work, we have determined and compared the intron/exon organization of the annelid genes for cytoplasmic GK, LK, AK, and mitochondrial TK and LK (MiTK and MiLK, respectively), and found that these annelid genes, irrespective of cytoplasmic or mitochondrial, have the same 8-intron/9-exon organization strikingly similar to mitochondrial CK (MiCK) genes. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. To gain a greater understanding of the evolutionary processes leading to the diversity of annelid phosphagen kinases, we determined for the first time the intron/exon organization of a cytoplasmic CK gene from a polychaete as well as that of another polychaete MiCK gene. These gene structures, coupled with a phylogenetic analyses of annelid enzymes and assessment of the fidelity of substrate specificity of some these phosphagen kinases, provide insight into the pattern of radiation of the annelid enzymes. Annelid phosphagen kinases appeared to have diverged in the following order (earliest first): (1) cytoplasmic AK, LK and TK, (2) GK, and (3) mitochondrial MiLK and MiTK. Interestingly, phylogenetic analyses showed that the above phosphagen kinases appear to be basal to all CK isoforms (mitochondrial, cytoplasmic and flagellar CKs). This somewhat paradoxical placement of CKs most likely reflects a higher rate of evolution and radiation of the annelid-specific LK, TK and GK genes than the CK isoform genes.     

Studies on phosphagen synthesis by mitochondrial preparations

Mitochondrial preparations from muscles of a crab (Cancer pagurus), two fish (Trachurus trachurus and Scyliorhinus canicula) and a bird (Columba livia) are able to synthesise, through ATP, the phosphagen related to that species. This indicates the presence of a bound phosphagen kinase. Addition of creatine kinase and creatine to crab mitochondria results in the synthesis of phosphocreatine. Similarly, the addition of arginine kinase and arginine to mitochondrial preparations from the fish and bird results in the synthesis of phosphoarginine. In the crab, the mitochondrial form of arginine kinase released by sonication had the same kinetic affinity constants and electrophoretic mobility and could not be distinguished immunologically from the cytosolic form. The close similarity of bound and cytosolic forms of arginine kinase in this crustacean suggests that the two forms have not evolved separately as has creatine kinase in the mammal.     

Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments.

The topology of the interfaces between actin monomers in microfilaments and three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase) was investigated using several specific antibodies directed against precisely located sequences in actin. A major contact area for glyceraldehyde-3-phosphate dehydrogenase was characterized in a region near residue 103. This interaction altered, by long-range conformational changes, the reactivity of antigenic epitopes in the C-terminal part of actin. The interface between actin and aldolase appeared to involve a sequence around residue 299 in the C-terminal region of actin. The interaction of phosphofructokinase, in contrast, modified the reactivity of all antibodies tested. Finally, the phosphagen kinases arginine kinase and creatine kinase showed no interaction with the microfilament.     

Energy transport and cell polarity: relationship of phosphagen kinase activity to sperm function

The energy required for motility of sea urchin sperm is transported from the mitochondrion to the flagellum by a phosphocreatine shuttle involving diffusion of phosphocreatine (PCr) between isozymes of creatine kinase (CrK) localized at the two sites (Tombes and Shapiro, Cell, 41:325, '85; Tombes et al., Biophys. J., 52:75, '87). The present studies demonstrate that high sperm CrK (various echinoderms; sea squirt, bristle worm, salmon) or arginine kinase (molusc, barnacle, moth) activity is seen in several species with sperm of a primitive morphology (mitochondrion at the base of the head, relatively long flagellum). In contrast, CrK activity is 10-100-fold less abundant in sperm of other species (frog, mouse, rooster, rabbit, bull, and human) that either possess a modified morphology (mitochondria that extend along the flagellum) and/or utilize glycolytic metabolism. We interpret these findings as support for the use of phosphagen kinase-dependent energy transport in cells in which the production of adenosine triphosphate (ATP) by the mitochondrion is distant from its utilization, leading to a form of metabolic polarization. Two other cell types, frog photoreceptors and rabbit oviduct cells, whose morphology and function also suggest that they exhibit metabolic polarization, contain relatively high CrK activity. The presence of high phosphagen kinase activity in metabolically polarized gametes and somatic cells further substantiates the role of such enzymes in facilitating energy transport.     

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.     

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.     

Arginine kinase: a common feature for management of energy reserves in African and American flagellated trypanosomatids

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.     

An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase

All phosphagen kinases contain a conserved cysteine residue which has been shown by crystallographic studies, on both creatine kinase and arginine kinase, to be located in the active site. There are conflicting reports as to whether this cysteine is essential for catalysis. In this study we have used site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine and methionine. In addition, we have replaced Cys282, conserved across all creatine kinases, with alanine. No activity was found with the C282M mutant. The C282S mutant showed significant, albeit greatly reduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation) reactions. The K(m) for creatine was increased approximately 10-fold, but the K(m) for phosphocreatine was relatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reported for rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/K(creatine) profile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolate anion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determined spectrophotometrically and found to be 5.6 +/- 0.1. The S284A mutant was found to have reduced catalytic activity, as well as a 15-fold increase in K(m) for creatine. The pK(a) of Cys282 in this mutant was found to be 6.7 +/- 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing to the unusually low pK(a) of Cys282.     

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.     

Conformational heterogeneity of creatine kinase determined from phase resolved fluorometry.

Fluorescence lifetimes of dimeric rabbit muscle creatine kinase specifically dansylated at both active sites and the homologous monomeric lobster muscle arginine kinase singly dansylated were determined using phase-modulation methods with global analysis of overdetermined data sets. For both proteins, the data is adequately described by three discrete exponential decays or a Lorentzian double distributed decay. Analogue phase resolved spectroscopy also reveals the presence of at least two distinct fluorophore domains for the dansyl moieties of creatine kinase. The model fluorophore, dansyllysine, exhibits a monoexponential decay with a value that is highly solvent dependent. Because the monomeric arginine kinase exhibits essentially the same decay law as doubly derivatized dimeric creatine kinase, it is proposed that the multiple lifetimes of creatine kinase reflect two or more isomeric dimeric states and not subunit asymmetry within a conformationally homogeneous dimeric population. Exposure of arginine kinase to 6 M guanidinium chloride results in a shift to shorter lifetimes and narrowing of the lifetime distributions. Creatine kinase displays a small narrowing of the distribution, but little change in fractional populations or lifetimes. These results suggest the presence of structural elements resistant to denaturation. The longest lifetime component in the triexponential discrete decay law of doubly dansylated creatine kinase is totally unquenched by acrylamide, whereas the two shorter lifetime components exhibit limited dynamic quenching. Steady-state quenching by acrylamide is significant and reveals a sharp distinction between accessible and nonaccessible dansyl groups. The major mechanism for interaction between the dansyl moieties and acrylamide is, atypically, static quenching.(ABSTRACT TRUNCATED AT 250 WORDS)