Effects of anions on a monomeric and a dimeric arginine kinase.

1. Some effects of anions on the rates of phosphoarginine synthesis by monomeric (lobster) and by dimeric (Holothuria forskali) arginine kinases are reported. 2. As with creatine kinase, acetate ions activate both enzymes: Cl- was also found to activate both although this was an inhibitor of creatine kinase. 3. NO3- inhibits the lobster enzyme. Inhibition is of the mixed type with respect to MgATP. Ki greater than Ki' and Ks greater than Ks' indicating that the presence of NO3- promotes the binding of substrate and vice versa. 4. NO3- alone has no effect on the difference spectrum of the lobster enzyme but in the presence of arginine, MgATP, MgADP, MgAMP or MgIDP the difference spectrum is greatly enhanced. A profound effect on the ionization state of tyrosine residues is inferred. 5. With the Holothuria enzyme low concentrations of NO3- activate in a manner that is competitive with arginine. Higher concentrations cause inhibition of the mixed type with respect to arginine in a similar manner to that found with MgATP for the lobster kinase. 6. Of a range of anions tested only NO3- and NO2- enhanced the inhibition of enzyme activity by MgADP, indicating the formation of a pseudo-transition-state dead-end complex, enzyme-arginine-NO3--MgADP. The effect was essentially independent of temperature with the Holothuria enzyme, but with the lobster enzyme was much less marked and temperature dependent. The difference may reflect the different stabilities of the monomer and dimer enzymes, although with neither arginine kinase is the stabilization of the dead-end complex as marked as is found with creatinine kinase.     

Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.     

Transient-phase studies on the arginine kinase reaction.

1. The initial formation of arginine phosphate by arginine kinase was studied in the time range 2.8--50 ms by the quenched-flow method. 2. A transient burst phase of product formation was obtained, the amplitude of which was temperature-dependent. At 35 degrees C it was 0.64 mol arginine phosphate/mol arginine kinase and at 12 degrees C, 0.25 mol/mol. 3. These results show that for the reaction pathway of arginine kinase the rate-limiting step follows the formation of arginine phosphate on the enzyme. This is in contrast to the creatine kinase reaction where no transient phase was observed [Engelborghs, Y., Marsh, A. & Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 4. The rate-limiting step on the arginine kinase reaction pathway is only slightly affected by temperature: the change in Kcat with temperature is due to a change of an equilibrium constant pertaining to at least two previous steps.     

Effect of substrate - binding on the immunologic reactivity of lobster - muscle arginine kinase : a comparison with rabbit - muscle creatine kinase.

The effects of substrate-binding upon the immunologic reactivity of rabbit creatine kinase and lobster arginine kinase have been investigated. The separate binding of the guanidine or the nucleotide substrate to creatine kinase yields no alteration of antigenicity and a substantial effect is only observed when all the loci at the active center of the enzyme, including that for the transferable phosphoryl group, are occupied. In contrast, the antigenic reactivity of arginine kinase is affected by the separate binding of either the guanidine or the nucleotide substrate, and the simultaneous binding of the two substrates results in a cumulative effect, which is irrespective of the phosphorylated or non-phosphorylated form of the complex. These results support the existence of substrate-induced conformational changes demonstrated by other methods, and they reveal appreciable differences in their effect on the antigenic reactivity of the two enzymes.     

Photoaffinity labelling of arginine kinase and creatine kinase with a gamma-P-substituted arylazido analogue of ATP

1. An ATP analogue with a photoactivated azide group attached to the gamma-phosphate via an amide bond, ATP gamma-p-azidoanilide, appeared to have potential use as a photoaffinity label for the nucleotide-binding regions of ATP: guanidine phosphotransferases. Upon photolysis in the presence of lobster muscle arginine kinase and rabbit muscle creatine kinase, the analogue is converted to a potent inhibito of these two kinases. This photo-dependent inhibition is specific as it cannot be induced by azidoaniline, a mixture of azidoaniline and ATP or by ATP gamma-p-aminoanilide. Preirradiated under suitable conditions, the photoanalogue still shows a transitory inhibitory effect which, however, slowly vanishes with time (t0.5 = 3 h). 2. The photoinhibition is significantly decreased by the presence of ATP or ADP but is completely prevented by the addition of a mixture of nucleotide and guanidine substrates. Differential spectroscopy and affinity chromatography on Sepharose-ATP demonstrated the inability of photoinactivated arginine kinase and creatine kinase to recognize their nucleotide substrates. 3. Experiments with [14C]ATP gamma-p-azidoanilide indicated that photolysis is associated with an irreversible and stoichiometric binding of the ATP analogue to the enzymes. Autoradiographs made with the peptide maps corresponding to the tryptic digests of each 14C-labelled photomodified enzyme showed an unexpected highly specific labelling of the proteins. 4. Thiiol titrations of the kinases which have been subjected to various photolysis conditions led to the conclusion that the arylnitrene moiety of the photoanalogue is covalently attached to the single reactive cysteinyl side chain present in the active-site region of the two homologous kinases. This amino acid residue appears, therefore, to be located near the phosphate chain binding subsite occupied by the ATP analogue and probably also by the natural nucleotide substrates.     

Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction.

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.     

The mechanism and modes of inhibition of arginine kinase from the cockroach (Periplaneta americana)

The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach (Periplanta americana) were investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for contraction and motility. Analysis reveals the following dissociation constants (mM) for the binary complex: E.Arg P-->E+Arg P, K=1.0; E.Arg-->E+Arg, K=0.45; E.MgATP-->E+MgATP, K=0.17; E.MgADP-->E+MgADP, K=0.12; and the ternary complex: Arg P.E.MgADP-->E.MgADP+Arg P, K=0.94; Arg.E.MgATP-->E.MgATP+Arg, K=0.49; MgATP.Enz.Arg-->E.Arg+MgATP, K=0.14; MgADP.E.Arg P-->E.Arg P+MgADP, K=0.09. For a particular substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating little, if any, cooperativity in substrate binding for the rapid equilibrium, random addition mechanism. The time course of the arginine kinase reaction exhibits a pronounced curvature, which, as described for enzyme from other sources, is attributed to formation of an inhibitory catalytic dead-end complex, MgADP.E.Arg. The curvature is accentuated by the addition of monovalent anions, including borate, thiocyanate, and, most notably, nitrite and nitrate. This effect is attributed to stabilization of the dead-end complex through formation of a transition state analog. However, the substantial decrease in initial velocity (92%) caused by nitrate is due to an additional inhibitory effect, further characterized as non-competitive inhibition (Ki=8.0 mM) with the substrate L-arginine. On the other hand, borate inhibition of the initial velocity is only 30% with significant subsequent curvature, suggesting that this anion functions as an inhibitor mainly by formation of a transition state analog. However, some component of the borate inhibition appears to be mediated by an apparent partial competitive inhibition with L-arginine. D-arginine is not a substrate for arginine kinase from the cockroach, but is an effective competitive inhibitor with a Ki=0.31 mM. L-Canavanine is a weak substrate for arginine kinase (Km=6.7 mM) with a Vmax for the pure enzyme that is approximately one-third that of L-arginine. However, initial velocity experiments of substrate mixtures suggest that competition between L-canavanine and L-arginine may not be a simple summation effect and may involve a structural modification. Sensitivity of arginine kinase activity to D-arginine as well as nitrate and borate anions, coupled with the fact that L-arginine is an essential amino acid for the cockroach, suggest that arginine kinase could be a useful chemotherapeutic target for the control of cockroach proliferation.     

Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen.

IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.     

Conformational changes in arginine kinase upon ligand binding seen by small-angle X-ray scattering

Small-angle X-ray scattering is used to study the effects of substrate binding to lobster arginine kinase in solution. We measure the radius of gyration of the enzyme in the absence and in the presence of ligands. We find that the radius of gyration decreases by 1.20 ± 0.25 Å upon binding ADP-Mg and L-arginine to form the ternary complex. The same decrease is also observed upon binding ADP-Mg alone or ATP-Mg. These results indicate a large conformational change consistent with the hinge motion of domains observed in other phosphokinases.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

Histidyls in lobster arginine kinase. 1H-NMR of native and ethoxyformylated enzyme, 1H- and 31P-NMR of its complexes with substrates and analogues

The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H-NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P-NMR spectroscopy of complexes with ADP. Five histidyls were detected by 1H-NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, shown from paramagnetic relaxation by Mn2+ to be close (less than or equal to 1.2 and less than or equal to 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO3- mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P-NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1. It is concluded that His-ethoxyformyl-enzyme is inactive because ethoxyformyl-His 1 is unable to titrate. This is consistent with His 1 acting as the acid-base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H-bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate.     

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.     

Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)

The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.     

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.     

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus.

1. Two molecular forms of arginine kinase, AK1 and AK2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK2 was retained on a DEAE-cellulose column at pH 7.5, but AK1 was not. 2. Both forms were monomeric (mol. wt. approximately 42,000) and showed the same pH optimum (7.5-8.0) in the direction of phosphoarginine synthesis. 3. AK1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower Km for L-arginine and higher Km for Mg(2+)-ATP than AK2. Unlike AK1, AK2 was strongly inhibited at high concentrations of Mg(2+)-ATP. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK1 showed a greater affinity than AK2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.     

Modification of arginine residues in pyruvate kinase (author's transl)

Pyruvate kinase from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. The loss of activity is caused by the reaction of a single molecule of phenylglyoxal per subunit of enzyme. During inactivation 3 - 6 arginyl residues are modified dependent on the concentration of phenylglyoxal used for modification. The solubility of the protein is reduced by the modification. ATP or phosphoenolpyruvate protect against inactivation. A single arginine is less subject to chemical modification in their presence. Therefore we assume that an arginine is essential at the substrate binding site. The activating ion K does not affectinactivation, where as Mg2 diminishes inactivation. Pyruvate kinase from rabbit muscle is modified by phenylglyoxal in a similar manner.     

Interaction in vitro of scallop muscle arginine kinase with filamentous actin.

Scallop muscle arginine kinase binds to F-actin from mollusc and rabbit muscle in vitro. One site of interaction appears to be located in residues 305-325 of a C-terminal fragment (residues 285-375) of actin. The binding is hindered in the presence of arginine, Mg(2+)-ADP and NO3-, which form a dead-end complex with the enzyme. F-actin inhibits the enzyme activity non-competitively with respect to Mg(2+)-ATP. As a function of arginine concentration, the inhibition is of the mixed type, where Km is affected more than Vmax.     

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.     

A direct continuous pH-spectrophotometric assay for arginine kinase activity

A direct and continuous spectrophotometric method was developed for determining arginine kinase (phosphoarginine synthesis) activity. Protons are produced during the phosphoarginine synthesis course, so adding the complex acid-base indicator to this solution and monitoring the decrease of absorbance of the solution at 575 nm will follow the arginine kinase activity. For this condition, one activity unit of arginine kinase was defined as 1 micromol H+ produced in 1 minute in the enzymatic reaction catalyzed by arginine kinase.     

Backbone resonance assignments of the 42 kDa enzyme arginine kinase in the transition state analogue form

Nearly complete backbone resonance assignments for the 357 residue, 42 kDa enzyme arginine kinase in a transition state analogue (TSA) complex are presented. The TSA is a quaternary complex of arginine kinase, MgADP, arginine, and nitrate. About 93 % (320 of 344) of the non-proline backbone amides were assigned using an enzyme enriched with ²H, ¹³C, and ¹⁵N in combination with three enzyme samples prepared with a single ¹⁵N-labeled amino acid (K, L, and R). The amide assignments will provide the foundation for investigating the dynamics of arginine kinase when in a TSA complex.