The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km^Tc and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.     

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases.

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.     

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.     

Membrane components can modulate the substrate specificity of protein kinase C

The cationic amphiphile, cholesteryl-3-carboxyamidoethylene-trimethylammonium iodide, can alter the substrate specificity of protein kinase C (PKC). The phosphorylation of histone catalyzed by PKC requires the binding of the enzyme to phospholipid vesicles. This cationic amphiphile reduces both the binding of PKC to lipid and as a consequence its rate of phosphorylation of histone. In contrast, PKC bound to large unilamellar vesicles (LUVs) composed of 50 mol % POPS, 20 mol % POPC, and 30 mol % of this amphiphile catalyzes protamine sulfate phosphorylation by an almost 4 fold greater rate. This activation requires phosphatidylserine (PS) and is inhibited by Ca²⁺. The extent of activation is affected by the time of incubation of PKC with LUVs. This data suggests a novel mechanism by which PKC-dependent signal transduction pathways may be altered by altering the protein targets of this enzyme.     

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 A with a final R of 22.3% (R(free) = 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.     

Screening of substrate analogs as potential enzyme inhibitors for the arginine kinase of Trypanosoma cruzi

Arginine kinase catalyzes the transphosphorylation between phosphoarginine and ADP. Phosphoarginine is involved in temporal ATP buffering and inorganic phosphate regulation. Trypanosoma cruzi arginine kinase phosphorylates only L-arginine (specific activity 398.9 x mUE-min(-1) x mg(-1)), and is inhibited by the arginine analogs, agmatine, canavanine, nitroarginine, and homoarginine. Canavanine and homoarginine also produce a significant inhibition of the epimastigote culture growth (79.7% and 55.8%, respectively). Inhibition constants were calculated for canavanine and homoarginine (7.55 and 6.02 mM, respectively). In addition, two novel guanidino kinase activities were detected in the epimastigote soluble extract. The development of the arginine kinase inhibitors of T. cruzi could be an important feature because the phosphagens biosynthetic pathway in trypanosomatids is different from the one in their mammalian hosts.     

Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6‐pentakisphosphate 2‐kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1‐Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110–140 within the N‐lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4‐, 5‐, and 6‐positions are recognized initially by the C‐lobe with subsequent interaction of the 1‐position phosphate by Arg130 that stabilizes this residue and the N‐lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N‐ and C‐lobes and kinase activation.     

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. Published in Russion in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1208–1214.     

Comparisons of creatine kinase primary structures

Comparisons of nine creatine kinase sequences show that 67% of the protein sequence is identical among rabbit, rat, mouse, and chicken muscle, rabbit, rat, and chicken brain, and electric organ sequences from two species of electric ray(Torpedo). The extensive homology precludes a facile prediction of active-site residues based on sequence conservation. The sequences are more similar within isozyme types than are the different isozymes from any one species. There are 35 positions in the muscle and brain sequence pairs for three species which differentiate the two forms. TheTorpedo sequences do not fall completely into either of these patterns. Except for homology with partial sequences of other ATP-guanidino phosphotransferases, no significant homology with other protein or nucleic acid sequences in available databases was found. Preliminary secondary structural predictions suggest that the C-terminal half of the protein is likely an /-type protein. Placement in the sequence of two peptides found in previous cross-linking studies reveals two stretches of primary structure that are presumably close in space to the reactive Cys-283 and hence close to the active site.     

Implications of the role of reactive cystein in arginine kinase: reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol

The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.     

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.     

The carp M1 muscle‐specific creatine kinase subisoform is adaptive to the synchronized changes in body temperature and intracellular pH that occur in the common carp Cyprinus carpio

The three previously cloned Cyprinus carpio muscle‐specific subisoforms of creatine kinase (CK, EC 2.7.3.2) designated M1‐, M2‐ and M3‐CK were examined. At temperatures <15° C and at pH >7·7, specific activities of M1‐CK were three to eight‐fold higher than specific activities of M3‐ and rabbit (R) M‐CK. At pH 8·0, M1‐CK exhibited its highest specific activity at 15° C. Michaelis constants of PCr () and ADP () of M1‐CK were relatively stable at pH between 7·1–8·0 and 25–5° C. Its calculated activation energy of catalysis (E_a) at pH 8·0 was lower than at pH 7·1. Circular dichroism spectroscopy results showed that changes in secondary structures in M1‐CK at the pH and temperatures studied were much less than in the cases of RM‐ and M3‐CK. The M1‐CK enzyme seemed to have evolved to adapt to the synchronized changes in body temperature and intracellular pH of C. carpio.     

The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.     

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates.     

Creatine metabolism and the consequences of creatine depletion in muscle

Currently, considerable research activities are focussing on biochemical, physiological and pathological aspects of the creatine kinase (CK) — phosphorylcreatine (PCr) — creatine (Cr) system (for reviews see [1, 2]), but only little effort is directed towards a thorough investigation of Cr metabolism as a whole. However, a detailed knowledge of Cr metabolism is essential for a deeper understanding of bioenergetics in general and, for example, of the effects of muscular dystrophies, atrophies, CK deficiencies (e.g. in transgenic animals) or Cr analogues on the energy metabolism of the tissues involved. Therefore, the present article provides a short overview on the reactions and enzymes involved in Cr biosynthesis and degradation, on the organization and regulation of Cr metabolism within the body, as well as on the metabolic consequences of 3-guanidinopropionate (GPA) feeding which is known to induce a Cr deficiency in muscle. In addition, the phenotype of muscles depleted of Cr and PCr by GPA feeding is put into context with recent investigations on the muscle phenotype of gene knockout mice deficient in the cytosolic muscle-type M-CK.Abbreviations Cr creatine - Crn creatinine - PCr phosphorylcreatine - CK creatine kinase - M-CK cytosolic muscle type CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - AGAT L-arginine: glycine amidinotransferase - GAMT S-adenosylmethionine: guanidinoacetate methyltransferase - Arg arginine - Met methionine - GPA guanidinopropionate=-guanidinopropionate - PGPA phosphorylated GPA - GBA 3-guanidinobutyrate=-guanidinobutyrate - CPEO chronic progressive external ophthalmoplegia     

Molecular characterization and kinetic properties of a novel two-domain taurocyamine kinase from the lung fluke Paragonimus westermani

Taurocyamine kinase (TK) was previously reported to be restricted to certain marine annelids; however, the present study has proven otherwise. The lung fluke Paragonimuswestermani has a contiguous two-domain TK with a mass of 80 216 Da consisting of 713 amino acid residues sharing higher sequence identity with molluscan arginine kinase (AK). Both domains of P. westermani TK have significant activity for the substrate taurocyamine and exhibited synergism during substrate binding. Since TK plays a key role in energy metabolism and is not present in mammals, inhibitors against P. westermani TK could be effective novel chemotherapeutic agents and could be utilized for the development of specific diagnostic tools for the detection of paragonimiasis.     

Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity

Human DDX3 (hDDX3) is a DEAD-box protein shown to possess RNA-unwinding and adenosine triphosphatase (ATPase) activities. The hDDX3 protein has been implicated in nuclear mRNA export, cell growth control, and cancer progression. In addition, a role of this protein in the replication of human immunodeficiency virus Type 1 and in the pathogenesis of hepatitis C virus has been recently proposed. Its enzymological properties, however, are largely unknown. In this work, we characterized its ATPase activity. We show that hDDX3 ATPase activity is stimulated by various ribo- and deoxynucleic acids. Comparative analysis with different nucleoside triphosphate analogs showed that the hDDX3 ATPase couples high catalytic efficiency to a rather relaxed substrate specificity, both in terms of base selection and sugar selection. In addition, its ability to recognize the L-stereoisomers of both 3' deoxy- and 2',3' dideoxy-ribose, points to a relaxed stereoselectivity. On the basis of these results, we hypothesize the presence of structural determinants on both the base and the sugar moieties, critical for nucleoside binding to the enzyme. Our results expand the knowledge about the DEAD-box RNA helicases in general and can be used for rational design of selective inhibitors of hDDX3, to be tested as potential antitumor and antiviral agents.