W. Ross Ellington

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.

Evolution of phosphagen kinase. VI. Isolation, characterization and cDNA-derived amino acid sequence of lombricine kinase from the earthworm Eisenia foetida , and identification of a possible candidate for the guanidine substrate recognition site

Lombricine kinase (LK) from the body wall muscle of the earthworm Eisenia foetida was purified to homogeneity. The enzyme was shown to be a dimer consisting of 40 kDa subunits. The cDNA-derived amino acid sequence of 370 residues of Eisenia LK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. This is the first reported lombricine kinase amino acid sequence. Alignment of Eisenia LK with those of creatine kinases (CKs), arginine kinases (AKs) and glycocyamine kinase (GK) suggested a region displaying remarkable amino acid deletions (referred to GS region), as a possible candidate for guanidine substrate recognition site. A phylogenetic analysis using amino acid sequences of all four phosphagen kinases indicates that CK, GK and LK probably evolved from a common immediate ancestor protein.

Induced fit in guanidino kinases - Comparison of substrate-free and transition state analog structures of arginine kinase

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate‐free) form. The final model has been refined at 2.35 Å with a final R of 22.3% (Rfree = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.

The role of phosphagen specificity loops in arginine kinase

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock‐and‐key mechanism. The second loop contacts the substrate with a valine in the methyl‐substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.

Energy metabolism during hypoxia in the isolated, perfused ventricle of the whelk,Busycon contrarium conrad

Energy metabolism and endogenous contractile activity during hypoxia were investigated in the isolated, perfused ventricle of the whelk,Busycon contrarium Conrad. Perfusion under hypoxic conditions for 2 h resulted in only small changes in contractile amplitude, but further perfusion resulted in variable responses ranging from no change to near cessation of contractile activity. The adenylate energy charge decreased only slightly after four hours of hypoxia. Contractile activity during hypoxia appears to be sustained by utilization of arginine phosphate and an activation of anaerobic energy metabolism. Alanine, succinate and octopine accumulated during hypoxia. Since aspartate levels decreased to one-third of the aerobic level while glutamate levels remained unchanged, it appears that aspartate provides the amino group in alanine formation. The results of the present study onB. contrarium ventricle support the hypothesis that glycogen and aspartate are simultaneously mobilized during the early phases of hypoxia and anoxia in this species.

Functional consequences of a gene duplication and fusion event in an arginine kinase

SUMMARY Arginine kinase (AK) from the foot of the razor clam Ensis directus consists of two full-length AK domains, denoted D1 and D2, fused in a single polypeptide chain. The full-length cDNA for Ensis AK was obtained and its deduced amino acid sequence was analyzed in the context of the X-ray crystal structure of a typical, monomeric AK. Both domains of Ensis AK contain most of the residues currently thought to be critical in catalysis, suggesting that both AK domains are catalytically competent. The full-length Ensis AK, a D2–NusA–His-tag fusion protein and a D2-truncated AK (enterokinase cleavage product of the fusion protein) were expressed in Escherichia coli and purified. All recombinant AK constructs displayed high enzyme activity. Attempts at expressing active D1 alone, D2 alone or a D1–NusA–His-tag fusion protein were unsuccessful. The catalytic properties of the active proteins were compared with the corresponding properties of recombinant AK from the horseshoe crab Limulus polyphemus, which is a typical monomeric AK. In contrast to expectations, the kinetic results strongly suggest that Ensis AK has only one active domain, namely D2. The Kcat values for all Ensis constructs were roughly twice that of typical AKs, indicating higher overall catalytic throughput at the competent active site. Furthermore, both the full-length and truncated D2 Ensis AKs showed no synergism of substrate binding unlike typical AKs. The D2–NusA–His-tag fusion construct actually displayed negative synergism of substrate binding, which means that, in effect, the first substrate bound acts as a competitive inhibitor of the second. The conservation of the structure of the apparently inactive D1 may be related to constraints imposed by structural changes that could potentially impact substrate binding in D2 and/or possibly influence the proper folding of the enzyme during synthesis. Overall, the results from the present study indicate that the AK contiguous dimer from Ensis directus functions with activity in only the second domain. Although lacking activity in D1, D2 appears to compensate by having a higher intrinsic catalytic throughput than typical 40-kDa monomeric AKs.

Induced Fit in Arginine Kinase

Creatine kinase (CK) and arginine kinase (AK) are related enzymes that reversibly transfer a phosphoryl group between a guanidino compound and ADP. In the buffering of ATP energy levels, they are central to energy metabolism and have been paradigms of classical enzymology. Comparison of the open substrate-free structure of CK and the closed substrate-bound structure of AK reveals differences that are consistent with prior biophysical evidence of substrate-induced conformational changes. Large and small domains undergo a hinged 13 degrees rotation. Several loops become ordered and adopt different positions in the presence of substrate, including one (residues 309-319) that moves 15 A to fold over the substrates. The conformational changes appear to be necessary in aligning the two substrates for catalysis, in configuring the active site only when productive phosphoryl transfer is possible, and excluding water from the active site to avoid wasteful ATP hydrolysis.

Origin of octameric creatine kinases

Mitochondrial creatine kinase (MiCK) occurs primarily as an octameric form localized in the mitochondrial intermembrane compartment in vertebrate tissues and echinoderm spermatozoa (both deuterostome groups). The octameric quaternary structure is thought to play important functional and enzyme targeting roles. We have found that the spermatozoa of the protostome polychaete Chaetopterus variopedatus contain three distinct isoenzymes of creatine kinase (CK) termed CK1, CK2 and CK3. CK3 appears to be present only in the sperm head/midpiece complex where mitochondria are restricted and has a subunit relative molecular mass (M r) of 43.4 kDa. Gel permeation chromatography using Superdex 200HR showed that CK3 has a native M r of 344.9 kDa indicating that this enzyme exists as an octamer. Electron micrographs of negatively stained CK3 preparations show structures which are virtually identical to those that have been seen for octameric vertebrate MiCK. The above observations show that CK3 from C. variopedatus displays great similarities to MiCKs from vertebrates and echinoderms. Octamerization of CK is not an advanced feature. The evolution of octameric subunit association is ancient and occurred prior to the divergence of protostomes and deuterostomes.

Phosphorus nuclear magnetic resonance studies of energy metabolism in molluscan tissues

SummaryIsolated ventricles of the whelkBusycon contrarium were perfused in 15 mm (internal diameter) nuclear magnetic resonance tubes at 22–24°C. Phosphorus nuclear magnetic resonance (31P-NMR) spectra were generated at 60.7071 MHz using a multi nuclei FT spectrometer. Methodologies were developed to utilize31P-NMR spectra to assess intracellular pH (pHi) and the relative levels of high energy phosphates in experimental preparations. Ventricles perfused under normoxic conditions for 6 h showed no changes in the levels of arginine phosphate and ATP. There was a slight decrease in pHi (0.08 unit). Anoxia and anoxia+ischemia resulted in a number of metabolic changes. There was a linear decay in arginine phosphate with half times of decay of 4.9 and 5.6 h, respectively. Inorganic phosphate levels rose 3–5 fold in both experimental groups. In contrast, no statistically significant changes in the adenylates were observed. 6 h of anoxia and anoxia+ischemia produced significant reductions in pHi. During anoxia, the pHi fell from 7.11 to 6.87, and during anoxia+ischemia the pHi fell from 7.14 to 6.79. Rates of accumulation of succinate and alanine under these conditions were quite low. The apparent low rates of glycolysis were probably related to minimal activities of phosphofructokinase due to reduced pHi and the lack of large alterations in the adenylates. The general response of the ventricle of the whelkB. contrarium to reduced oxygen tensions is a reduction in energy demands leading to low rates of anaerobic energy metabolism and resultant alterations in pHi and levels of high energy phosphates.

Evolution and divergence of the genes for cytoplasmic, mitochondrial, and flagellar creatine kinases.

Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intracellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodimers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet another gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in protochordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a protochordate tunicate. These new sequences, coupled with available sequences in the databases and sequences extracted from genome sequencing projects, provide revealing insights into the evolution and divergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved after the divergence of the craniates from the protochordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in structure consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flagellar CK genes are rather ancient and that there has been a systematic pattern of duplication and divergence consistent with changing nature of energy demands and physicochemical environment in the cells where they are expressed.