Conformational change and inactivation of arginine kinase from shrimp Feneropenaeus chinensis in oxidized dithiothreitol solutions.

The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS-PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.     

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 active site cysteine of arginine kinase: structural and functional analysis of partially active mutants

Arginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady-state binding constants were slightly increased, more so for phospho-L-arginine than ADP. Substrate binding synergy observed in many phosphagen kinases was reduced or eliminated in mutant enzymes. The crystallographic structure of the alanine mutant at 2.3 A resolution, determined as a transition state analogue complex with arginine, nitrate, and MgADP, was nearly identical to wild type. Enzyme-substrate interactions are maintained as in wild type, and substrates remain at least roughly aligned for in-line phosphoryl transfer. Homology models with serine, asparagine, or aspartate replacing the active site cysteine similarly show only minor structural changes. Most striking, however, is the presence in the C271A mutant crystallographic structure of a chloride ion within 3.5 A of the nonreactive N(eta) substrate nitrogen, approximating the position of the sulfur in the wild-type's cysteine. Together, the results contradict prevailing speculation that the cysteine mediates a substrate-induced conformational change, confirm that it is the thiolate form that is relevant to catalysis, and suggest that one of its roles is to help to enhance the catalytic rate through electrostatic stabilization of the transition state.     

Structural interpretation of reduced insulin activity as seen in the crystal structure of human Arg-insulin

The N-terminal glycine of the A-chain in insulin is reported to be one of the residues that binds to the insulin receptor. Modifications near this region lead to variations in the biological activity of insulin. One such modification viz., an addition of an arginine at the N-terminal A-chain, was reported to possess two-thirds the activity of native insulin. The crystal structure of 2 zinc human arg (A0) insulin has been elucidated to 2A resolution to understand the mechanism of reduction in insulin activity. A conformational transition from T6 to T3R3(f) and a decrease in the surface accessibility of residues in the so called receptor binding region have been observed. The presence of arginine has also induced distortions in the A chain N-terminal helix. The subtle conformational alterations like decrease in surface accessibility, alterations in the charge surface and changes in the relative orientation of the two helices in the A chain may be responsible for the reduction in activity.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。     

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0–2.8 M in the presence of camphor and 0–1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20–60 °C in the presence of the substrate and 20–47 °C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.     

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.     

Effects of aspartic acid and potassium chloride on arginine kinase from shrimp

The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.     

Accelerating effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein

The effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine and human plasmas. The sulfatide-mediated activation of factor XII and prekallikrein in the presence of high-molecular-weight (HMW) kininogen was remarkably accelerated by 10(-5) M zinc ions. This accelerating effect was observed only in the presence of HMW kininogen. The kinetic analysis of the accelerating effect of zinc ions demonstrated that zinc ions reduce the Km values and increase the Vmax values on the activation of factor XII by kallikrein and on the activation of prekallikrein by factor XIIa. The value of Vmax/Km increased 26.4-fold in the former reaction and 2.8-fold in the latter reaction, indicating that zinc ions accelerate mainly the activation of factor XII by kallikrein. In the presence of 5 x 10(-4) M zinc ions, typical difference spectra due to a red shift of tryptophan and/or tyrosine residues were observed for HMW kininogen and its derivatives but not low-molecular-weight (LMW) kininogen. Since the concentration of zinc ions required to induce the difference spectra is comparable with that to enhance the activation of factor XII and prekallikrein, it appears that there is some correlation between the conformational change of HMW kininogen and the enhancement of the activation.     

The interaction between residues 62 and 193 play a key role in activity and structural stability of arginine kinase

The purpose of this study is to clarify that the amino acid residues (Asp62 and Arg193) are responsible for the activity and stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. In this research, we replaced D62 with glutamate (E) or glycine (G) and R193 with lysine (K) or glycine (G). The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of ultraviolet (UV) difference and fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase from greasyback shrimp (Metapenaeus ensis).     

Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate

Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn2(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn2(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn2(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn2(+) on metabolic enzymes in marine invertebrates.     

Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in alphaB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of alpha-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of alpha-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of alpha-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of alpha-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.     

Inactivation and conformational changes of creatine kinase at low concentrations of hexafluoroisopropanol solutions.

Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%-5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%-2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate-enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole.     

Utility of arginine kinase for resolution of phylogenetic relationships among Brachyuran genera and families

The molecular phylogenetics of decapod crustaceans has been based on sequence data from a limited number of genes. These have included rapidly evolving mitochondrial genes, which are most appropriate for studies of closely related species, and slowly evolving nuclear ribosomal RNA genes, which have been most useful for resolution of deep branches within the Decapoda. Here we examine the utility of the nuclear gene that encodes arginine kinase for phylogenetic reconstruction at intermediate levels (relationships among genera and families) within the decapod infraorder Brachyura (the true crabs). Analyses based on arginine kinase sequences were compared and combined with those for the mitochondrial cytochrome oxidase I gene. All of the genera in our taxon sample were resolved with high support with arginine kinase data alone. However, some of these genera were grouped into clades that are in conflict with recognized brachyuran families. A phylogeny based on cytochrome oxidase I was consistent with the arginine kinase phylogeny, but with weaker support. A recently proposed measure of phylogenetic informativeness indicated that arginine kinase was generally more informative than cytochrome oxidase I for relationships above the level of genus. Combined analysis of data from both genes provided strong support for clades that are in conflict with current assignments of genera to the families Epialtidae, Mithracidae, Pisidae, and Portunidae.     

Alkaline unfolding and salt-induced folding of arginine kinase from shrimp Feneropenaeus chinensis under high pH conditions

The structural and functional properties of arginine kinase (AK) in alkaline conditions in the absence or presence of salt have been investigated. The conformational changes of AK during alkaline unfolding and salt-induced folding at alkaline pH were monitored using intrinsic fluorescence emission, binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate and circular dichroism. The results for the alkaline unfolded enzyme showed that much lower pH (11.0) was required to cause the complete loss of AK activity than was required to cause an obvious conformational change of the enzyme. Compared with the completely unfolded state in 5 M urea, the high pH denatured enzyme had some residual secondary and tertiary structure even at pH 13.0. Increasing the ionic strength by adding salt at pH 12.75 resulted in the formation of a relatively compact tertiary structure and a little new secondary structure with hydrophobic surface enhancement. These results indicate that the partially folded state formed under alkaline conditions may have similarities to the molten globule state which is compact, but it has a poorly defined tertiary structure and a native-like secondary structure.     

Rate-limiting domain and loop motions in arginine kinase

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and arginine. It is the arthropod homologue of creatine kinase, buffering cellular ATP levels. Crystal structures of arginine kinase, in substrate-free and substrate-bound forms, have revealed large conformational changes associated with the catalytic cycle. Recent nuclear magnetic resonance identified movements of the N-terminal domain and a loop comprising residues I182--G209 with conformational exchange rates in the substrate-free enzyme similar to the turnover rate. Here, to understand whether these motions might be rate-limiting, we determined activation barriers for both the intrinsic dynamics and enzyme turnover using measurements over a temperature range of 15-30 °C. (15)N transverse relaxation dispersion yields activation barriers of 46 ± 8 and 34 ± 12 kJ/mol for the N-terminal domain and I182--G209 loop, respectively. An activation barrier of 34 ± 13 kJ/mol was obtained for enzyme turnover from steady-state kinetics. The similarity between the activation barriers is indeed consistent with turnover being limited by backbone conformational dynamics and pinpoints the locations of potentially rate-limiting motions.