Hypotaurocyamine kinase evolved from a gene for arginine kinase

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

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

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

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu³¹⁴ decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe³¹⁵ mutants including the AAloop which retained Glu³¹⁴ had modest AK activity (5–84% of the wild type). All the results above suggested that Glu³¹⁴ played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu³¹⁴ mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe³¹⁵ in dimeric AK has a different role from His³¹⁵ in monomeric AK. This might contribute to the stabilization of the native conformation, while His³¹⁵ in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.     

Enzymatic cycling method using creatine kinase to measure creatine by real-time detection

We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP–GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP–GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

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.     

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Early gene interaction during prepupal expression of Drosophila arginine kinase

Arginine kinase displays a distinctive rise and fall in specific activity and specific protein levels during the prepupal stage of Drosophila development with maximal activity occurring at morphological stage P3. This developmentally regulated peak is under the influence of ecdysone. Altered doses of the major ecdysone-inducible “early” genes at cytological regions 75B and 2B5 alter this pattern of expression while altered doses of another major “early” gene at 74EF have no effect. We hypothesize that a product of the 2B5 locus and a product of the 75B locus interact to effect this developmental pattern of expression of Drosophila arginine kinase. © 1992 Wiley-Liss, Inc.     

Mitochondrial creatine kinase with atypical pI values detected in serum of a patient with ovarian hepatoid yolk sac tumor

Atypical mitochondrial creatine kinase (creatine N-phosphotransferase, CK, EC 2.7.3.2) was detected in the serum of a patient with carcinoma of germ cell origin, probably hepatoid yolk sac tumor. The pI of the oligomeric atypical mitochondrial CK (Mi-CK) was found at the acidic side compared to that of the typical ubiquitous Mi-CK (uMi-CK), while the molecular size of the atypical Mi-CK was similar to that of the typical uMi-CK. The pIs of the oligomeric and the dimeric atypical Mi-CKs became the same as those of the typical uMi-CK upon treatment with 2-mercaptoethanol. Therefore, the atypical Mi-CK was suggested to be an oxidized form of uMi-CK, and the oxidation might have occurred in the mitochondria because the oligomeric atypical Mi-CK had atypical pIs. The physicochemical characteristics of the oxidized uMi-CK were similar to those of the typical uMi-CK.     

Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg²⁺ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg²⁺ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg²⁺; however, the rates of reactivation are independent of the Mg²⁺ concentration. In addition, Mg²⁺ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg²⁺ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg²⁺ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.     

Cyclic peptides containing tryptophan and arginine as Src kinase inhibitors

A number of cyclic and linear peptides containing various combinations of amino acids were evaluated for their Src kinase inhibitory potency. Among all the peptides, cyclic decapeptide C[RW]₅ containing alternative arginine (R) and tryptophan (W) residues was found to be the most potent Src kinase inhibitor. C[RW]₅ showed higher inhibitory activity (IC₅₀ = 2.8 μM) than C[KW]₅, L(KW)₅, C[RW]₄, and C[RW]₃ with IC₅₀ values of 46.9, 69.1, 21.5, and 25.0 μM, respectively, as determined in a fluorescence intensity-based assay. Thus, the cyclic nature, the presence of arginine, ring size, and the number of amino acids in the structure of the peptide were found to be critical in Src kinase inhibitory potency. The IC₅₀ value of C[RW]₅ was found to be 0.8 μM in a radioactive assay using [γ-³²P]-ATP and polyE₄Y as the substrate. C[RW]₅ was a noncompetitive Src kinase inhibitor, showing approximately fourfold more selectivity towards Src than Abl.     

Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site

Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (ΔH°) was 12.38 kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK.     

Reactivation and refolding of reassociated dimers of rabbit muscle creatine kinase

Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k ₁ = 4.88 × 10^–3 s^–1, k ₂ = 0.68 × 10^–3 s^–1). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k ₁ = 3.28 × 10^–3 s^–1, k ₂ = 0.11 × 10^–3 s ^–1) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

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

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.