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Val65 plays an important role in the substrate synergism, structural stability and activity of arginine kinase
International Journal of Biological Macromolecules 45 (2009) 393–398
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Val65 plays an important role in the substrate synergism, structural stability and activity of arginine kinase Qing-Yun Wu a,c,1 , Feng Li a,1 , Xiao-Yun Wang a,b,∗ a b c
College of Life Science, Shandong Agricultural University, Tai’an, Shandong 271018, People’s Republic of China State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, People’s Republic of China Institutes of Biochemistry and Cell Biology, State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 22 May 2009 Received in revised form 29 June 2009 Accepted 30 June 2009 Available online 21 July 2009 Keywords: Arginine kinase Mutagenesis Structural stability Substrate synergism Activity
a b s t r a c t Arginine kinase, a member of phosphagen kinase, is a key enzyme in the cellular energy metabolism of invertebrates. A series mutation of conserved amino acid residue V65 was constructed to investigate its role in AK substrate synergism, structural stability and activity. Our study revealed that mutation in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results provided herein suggest that amino acid residue V65 played a relatively important role in AK substrate synergism, structural stability and activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Arginine kinase (ATP: l-arginine phosphotransferase EC 2.7.3.3) (AK) is a member of what appears to be a highly conserved family of phosphotransferases that reversibly catalyze the transfer of phosphate from phosphoarginine to ADP, yielding ATP [1]. Both of these enzymes play an important role in cells, by buffering the ATP concentration according to cellular energy requirements [2–5]. Recently, a crystal structure for the transition state analog complex (TSAC) of the AK from the horseshoe crab Limulus has appeared [6]. Substrate binding in both CK and AK involves pronounced conformational changes [7–9], in which the two flexible loops move in such away as to clamp down on the substrates [7]. Although induced fit occurred in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be complicated [7–9]. The structure showed that four amino acid residues (Ser63, Gly64, Val65 and Tyr68 in Limulus AK sequence),
Abbreviations: AK, arginine kinase; CK, creatine kinase; IPTG, isopropyld-thiogalactopyranoside; ANS, 1-anilinonaphtalene-8-sulfonate; TSAC, transition state analog complex; GdnHCl, guanidine hydrochloride; Emax, emission maximum wavelength of the intrinsic fluorescence. ∗ Corresponding author at: College of Life Science, Shandong Agricultural University, Dai zong 61, Tai’an, Shandong 271018, People’s Republic of China. Tel.: +86 538 8242656 8430; fax: +86 538 8242217. E-mail address: [email protected] (X.-Y. Wang). 1 These authors contributed equally to this work. 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.06.016
located in the guanidine specificity region (GS region), might form hydrogen bond with the substrate arginine. This GS region was overlapped partly by the so-called flexible loop in the crystal structures of chicken mitochondrial CK [10] and Limulus AK [6]. There was a proportional relationship between the size of the deletion in the GS region and the mass of guanidine substrate used [11]. The amino acid residue Tyr68 was conserved in all AKs, except in the sea cucumber Stichopus AK that evolved from the CK gene, which suggested that the Tyr68 appeared to be the most important residue in substrate binding [12]. The amino acid residues Ser63, Gly64 were also reported to play essential roles in the substrate binding [13]. Alignment of amino acid sequences indicates that Val65 amino acid residue is conserved in all of guanidino kinases. Moreover, the structure analysis suggested that the amino acid residue Val65 might form a hydrogen bond with the amino group of the substrate arginine in Limulus AK, and we assumed that this residue might play a relative important role in substrate binding. However, little is known about the role of this amino acid residue Val65 in keeping AK structural stability, substrate synergism and activity. In our previous study, we demonstrated that some amino acid residues outside the active site played important roles in AK activity, substrate synergism and structural stability [14–16]. Does the amino acid residue V65 mutation affect AK function. In this study, the typical nonpolar amino acid valine, V65, was mutated to the non-typical nonpolar, uncharged and negatively charged amino acids of Gly (G), Ser (S) and Asp (D), respectively, to investigate its role in AK substrate synergism, structural stability and activity. The results suggested that this conserved residue may play an
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important role in AK substrate synergism, structural stability and activity. 2. Materials and methods 2.1. Cloning, site-directed mutagenesis and expression of the mutant locust AK pMD-18T plasmid with locust AK cDNA (pMD-Locust WT-AK) inserted was used as a template for mutagenesis [17]. The gene encodes 355 amino acids with a molecular weight of about 40 kDa. Five mutations (V65G, V65S, V65D, V65A and V65L) were introduced into the template of the pMD-locust WT-AK by overlap PCR using mutation primers. The sequences of these mutation primers for these mutations were as follows: for V65G, 5 CATGACTCTGGTGGTGGTATCTATGC-3 and 5 -GCATAGATACCACCACC AGAGTCATG-3 ; for V65S, 5 -ATGACTCTGGTTCTGGTATCTATGC-3 and 5 -GCATAGATACCAGAACCAGAGTCAT-3 ; for V65D, 5 CATGACTCTGGTGAC GGTATCTATGC-3 and 5 -GCATAGATACCGTCACCAGAGTCATG-3 , for V65A, 5 -CATGACTCTGGTGCTGGTATCTATGC-3 and 5 -GCATAGATACCAGCACCAGAG TCATG-3 , for V65L, 5 CATGACTCTGGTCTGGGTATCTATGC-3 and 5 -GCATA GATACCCAGACCAGA GTCATG-3 , mutated sequences were underlined. The PCR primers for the full-length gene were 5 -GATGGATCCATGGTTGATGCTGCAGTGCT G-3 and 5 -GACAAGCTTCAGAGTGCCTTACAGAGTCC-3 , in which the restriction sites introduced into the primers were underlined. Finally, the mutant products were completely sequenced to confirm that only the intended mutations had been introduced. The mutant gene was cloned into expression vector pET-28a and transformed into the Escherichia coli BL21 (DE3) codon plus. The His6 -tagged locust AK fusion protein was expressed in E. coli BL21 by induction with 2 mM isopropyl-1-thio--dgalactopyranoside (IPTG) at 25 ◦ C for 14 h. The soluble protein was extracted and purified using a nickel affinity chromatography column purification kit. The purity was checked by SDS-PAGE.
glycine–NaOH, 1 mM DTT at pH 8.6) for 24 h at equilibrium state. The refolding experiment was initiated by dilution of the denatured AKs into the standard buffer (pH 8.6) to final GdnHCl concentrations ranging from 0.1 M to 4 M at 25 ◦ C. The intrinsic and ANS fluorescence spectra of unfolding and refolding AKs were collected on an F-4500 spectrofluorometer using a 1-cm path-length cuvette being two Trp residues (position 214 and 221) in AK. 2.5. Parameter A and phase diagram analysis of intrinsic fluorescence data To more clearly characterize the effects of mutations on AK folding, Parameter A and phase diagram analysis, which were sensitive tools to identify folding intermediates, were used to compare the folding pathways of the WT and mutant AKs. Parameter A, which reflected the spectral shape of the intrinsic Trp fluorescence [21], was obtained by dividing the fluorescence intensity at 320 nm (I320 ) by the intensity at 365 nm (I365 ) during unfolding and refolding. The “phase diagram” analysis was carried out as described previously [22]. The fluorescence data were normalized by I320 and I365 of the spectra during unfolding and refolding. In the phase diagram, the joint position of two lines indicated that an intermediate appeared at the corresponding GdnHCl concentration. 3. Results 3.1. Expression of the mutant locust AK and enzymatic analysis All of the recombinant enzymes were successfully expressed as soluble fusion proteins with His6 -tag, and purified by affinity chromatography and size exclusion chromatography. The enzymatic characteristics of the recombinant wild-type (WT) fusion enzymes were very similar to that of native locust AK, indicating the Nterminal His6 -tag portion had no effect on their activity. 3.2. Kinetic parameters analysis
2.2. Enzyme assay and determination of kinetic parameters AK activity (phosphoarginine synthesis) was assayed using a modification of previous procedures [18,19]. The assay mixture for AK activity determination consisted of 100 mM Tris (pH 8.0), 10 mM l-arginine, 2 mM ATP-Na, 10 mM mercaptoethanol and 10 L of 0.01 mM enzyme solution. The absorbance at 660 nm was measured at 30 ◦ C using an Ultrospec4300 pro UV-vis Spectrophotometer. Protein concentration was determined by Bradford’ method [20], using an Ultrospec4300 pro UV-vis Spectrophotometer. The two-substrate graphical method was used to obtain the kinetic parameters [14]. The enzyme assays were carried out at the optimum pH (pH 8.6) and temperature (30 ◦ C) with different concentrations of ATP and arginine. And all these reactions were carried out at least four times. 2.3. Thermal stability of AK The thermal stability of WT and mutants AKs was determined by activity assay after incubation at different temperatures. The enzyme solutions were incubated at given temperatures varying from 25 ◦ C to 65 ◦ C for 10 min, then cooled on ice and the activity was measured at 30 ◦ C. The aggregation of AKs at given temperature was monitored by measuring the turbidity at 400 nm. 2.4. Unfolding and refolding experiments The native and mutant AKs were added to different concentrations of GdnHCl dissolved in the standard buffer (10 mM
Table 1 shows the effects of mutations of V65 on AK enzyme kinetics. The catalytic constants of mutations decreased in different degrees, about 86.7%, 73.5%, 62.6%, 57.3% and 33.4% of V65G, V65A, V65L, V65S and V65D compared with that of WT-AK, respectively. It shows when the typical nonpolar amino acid V65 was replaced by polar amino acids (V65D and V65S), the enzyme activities decreased most. Similarly, the binding affinity of arginine and ATP in the mutant AKs of polar amino acids was lower than nonpolar mutations, compared to that of WT-AK, as indicated by about 2- to 3-folds increase of the Km values for ATP and arginine. However, the Kd values for arginine and ATP of these mutants were increased a little, about 1.14- to 1.37-folds compared with that of WT-AK, resulting in a Kd /Km value of 1.08–1.56, indicating that the synergism in substrate binding of WT-AK (Kd /Km value 2.80) was decreased. More importantly, the substrate synergism for the mutation V65D Arg had almost lost and its kcat /Km value (18.94 s−1 mM−1 ) showed 9-folds decline in enzyme catalysis efficiency, compared with that (167.6 s−1 mM−1 ) of WT-AK. When V65 was replaced by nonpolar amino acids (V65G, V65A and V65L), the synergism in substrate binding also decreased, less than that of polar amino acids, Kd /Km value between 1.91 and 2.46. The changes of synergism may be caused by polarity of the amino acids. To sum up, when V65 was replaced by the nonpolar amino acids it led to moderate changes, while when it was replaced by the polar amino acid (uncharged S or the negatively charged D), it led to significant changes in the substrate binding and synergism. Glycine is special in polarity, between nonpolar and polar amino acids. The replacement of the residues in the mutants (D and S) might lead to the much looser tertiary
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Table 1 Comparison of kinetic parameters for the forward reaction of recombinant WT and mutant AKs. Vmax , mol Pi min−1 mg−1
Mutants a
Recombinant WT AK V65G V65S V65D V65A V65L
239.1 208.5 136.8 78.2 176.3 149.9
± ± ± ± ± ±
7.65 3.61 3.11 3.04 4.04 3.66
Arg
kcat , s−1 159.4 138.4 91.3 53.2 117.2 99.8
± ± ± ± ± ±
Km , mM 6.2 1.81 2.11 1.91 1.82 1.69
0.951 1.48 2.15 2.81 1.69 1.85
± ± ± ± ± ±
0.08 0.15 0.23 0.31 0.21 0.19
Arg
Kd , mM 2.67 3.64 3.35 3.04 3.62 3.53
± ± ± ± ± ±
0.22 1.05 0.72 0.51 0.99 1.08
ATP Km , mM
1.270 2.028 3.11 3.71 2.26 2.47
± ± ± ± ± ±
0.23 0.52 0.44 0.75 1.05 1.02
Arg
Kd
,
3.56 5.13 4.85 4.01 4.93 4.72
Arg
mM
kcat /Km , s−1 mM
± ± ± ± ± ±
167.6 93.51 42.5 18.9 69.4 53.9
0.32 1.84 1.13 0.54 1.77 1.66
± ± ± ± ± ±
Kd /Km
5.34 4.36 3.31 0.89 2.98 2.53
2.80 2.46 1.56 1.08 2.18 1.91
± ± ± ± ± ±
0.66 0.99 0.24 0.36 0.78 0.64
Note: Kinetic parameters were obtained from at least three runs of the reaction. a Kinetic parameters cited from the article [14].
structures than that of WT-AK. Now that this amino acid did not participate in the catalytic mechanism of AK and the substrate binding of ATP, the effects of the mutation on the substrate binding affinity of ATP and the substrate synergism of AK might be due to the impaired tertiary structure of AK. 3.3. Effects of mutations on AK structure Concerning the tertiary structure, the emission maximum of the intrinsic fluorescence (Fig. 1A) was found to red shift to about 333 nm for V65G, about 336 nm for V65S and about 338 nm for V65D compared to the WT-AK (331 nm), which suggested that the Trp residues in mutant AKs were more exposed to water and the tertiary structures of the mutations were much looser than that of WT-AK. The fluorescence spectrum of V65A and V65L were almost identical with WT-AK (data not shown). The ANS fluorescence intensity of all mutations was higher than that of WT-AK at the same concentration (Fig. 1B) which reflected that the mutant AKs had more hydrophobic exposure than that of WT-AK to allow the binding of the ANS molecules. These spectroscopic experiments clearly indicated that the mutations impaired the tertiary structures of AK. Analyzing the reason, the replacement of the hydrophobic by hydrophilic amino acid would weaken the hydrophobic interactions which may essential in sustaining AK structure stability and finally result in the more hydrophobic exposure and the much looser tertiary structures. 3.4. Effects of mutations on AK thermal stability and thermal aggregation The effects of mutations on AK stability were evaluated by the residual activity retained after 10 min heat treatment (Fig. 2A). The activity of WT-AK had few changes after heat treatment at temperatures below 40 ◦ C. A steep decrease of activity was observed
between 40 ◦ C and 60 ◦ C, and a complete loss in activity occurred above 65 ◦ C. All the mutations were much less stable than that of WT-AK. The midpoints of thermal inactivation of WT-AK and the mutations V65G, V65A, V65L, V65S and V65D were about 53.6 ± 0.5 ◦ C, 49.4 ± 0.5 ◦ C, 50.2 ± 0.5 ◦ C, 48.3 ± 0.5 ◦ C, 43.6 ± 0.5 ◦ C and 39.3 ± 0.5 ◦ C, respectively. The aggregation experiment also showed the similar effects of mutations on AK conformation. Only a little aggregation occurred after heated at 41 ◦ C for 60 min for WT-AK, whereas considerable aggregation was observed for all mutations at 45 ◦ C (Fig. 2B). When the heating temperature was increased to 54 ◦ C, the aggregation rate of mutations was much faster than that of WT-AK. Combining the above results, we can deduce that in the mutations the impaired structure and greater hydrophobic exposure might facilitate protein aggregation at elevated temperatures because the hydrophobic amino acid was replaced by the hydrophilic amino acid. 3.5. Effects of the mutations on AK unfolding and refolding Spectroscopic methods were used to investigate the effects of mutations on AK unfolding and refolding. As shown in Fig. 3A and B, in the unfolding and refolding process, increasing or decreasing GdnHCl concentrations caused a sharp red or blue shift of the emission maximum. However, in the ranges of 0.2–0.5 M and 0.9–1.2 M GdnHCl, there was little change in the emission maximum for the mutant, while that of WT-AK was 0.3–0.6 M and 0.9–1.2 M GdnHCl. Meanwhile, the ANS intensity reached a maximum at 0.3 M and 1.0 M GdnHCl for the mutations, while that of the WT-AK was at 0.4 M and 1.0 M GdnHCl, as was shown in Fig. 3C and D. (The results of V65A and V65L were almost identical with that of V65G, so only V65G was shown here.) These results implied that two equilibrium intermediate states existed in the folding process of WT and mutant AKs although there was little different in concentration
Fig. 1. Effects of mutations on AK tertiary structures, intrinsic fluorescence spectra (A), and ANS fluorescence spectra (B). Curve (1) represents spectra of WT-AK, (2) V65G, (3) V65S, (4) V65D, and (5) represents the emission fluorescence spectra of ANS solution. The WT and mutant AK was dissolved in standard buffer at a final concentration of 3.2 M. ANS fluorescence spectra were recorded using an excitation wavelength of 380 nm.
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Fig. 2. Thermal stability and aggregation of AKs. (A) Thermal stability of AKs. (B) Thermal aggregation of AKs at 45 ◦ C.
of GdnHCl that the equilibrium intermediate states formation. The first intermediate was very similar to that of the pre-molten globule state with no activity, and the second has the characteristics of the highly ordered molten globule state with partial activity, which was quite consistent with previous observations [22,23]. The transitions of WT and mutant AKs refolding from the GdnHCl-denatured state were similar to those unfolding at GdnHCl concentrations above 0.5 M when monitored by these two techniques. The deviation between the unfolding and refolding at GdnHCl concentrations below 0.5 M indicated that the folding of WT and mutant AKs was not fully reversible under our conditions. Nevertheless, the transition curves of the mutant AKs were almost identical to that of WT-AK at GdnHCl concentrations above 0.5 M, which suggested that the mutation did not affect the M↔G↔U transitions. The major
difference in folding between WT-AK and mutations was observed at low GdnHCl concentrations. All these results suggested that the major differences in folding between WT-AK and mutations at low GdnHCl concentrations might be caused by the differences in their native structures (Fig. 1). Parameter A and phase diagram analysis were also used to clarify the effects of mutations on AK folding. As presented in Fig. 4A and B, the unfolding transition curves of mutations V65S, V65D and V65G were almost identical to WT-AK at all conditions for GdnHCl concentrations varying from 0.5 M to 6 M. Furthermore, as indicated by the phase diagram analysis in Fig. 4C and D, all the transitions could be fitted by three linear parts, which suggested that all the transitions were a three independent transitions process with the appearance of two intermediates although the GdnHCl concentra-
Fig. 3. Unfolding and refolding of WT and mutant AKs, maximum emission wavelength (Emax ) of the intrinsic fluorescence (A) and the intensity of ANS fluorescence (C) in unfolding, maximum emission wavelength (Emax ) of the intrinsic fluorescence (B) and the intensity of ANS fluorescence (D) in refolding. The final concentration of AK was 3.2 M.
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Fig. 4. Parameter A (A–C) and phase diagram (D–F) analysis of intrinsic fluorescence data in unfolding. Parameter A was obtained by dividing the fluorescence intensity at 320 nm (I320 ) by the intensity at 365 nm (I365 ). The phase diagram was constructed by I320 vs. I365 for the unfolding of AK.
tion of the appearance of the first intermediates of the mutant (0.3 M) was different from that of WT-AK (0.4 M). The joint position of the two lines above 0.5 M was also at the same GdnHCl concentration, when below the 0.5 M the joint position of the two lines was at 0.3 M for the mutant, while that of the WT-AK was 0.4 M. In the refolding experiments, the refolding transition curves of all the mutations showed the same results (data not shown). These results coincided with those from direct spectra analysis in Fig. 3, which also suggested that the mutation did not affect the folding pathways of AK. The discrepancy between the WT and the mutant unfolding at low concentrations of GdnHCl was more likely to be caused by their different native structures, but not by the interference of the folding pathways by the mutation. 4. Discussion Phosphagen (guanidino) kinases contain a family of highly conserved enzymes, which catalyze the reversible transfer of phos-
phate from phosphagen such as creatine phosphate to ADP. AK similar to CK in vertebrates is a phosphagen kinase participating in cell metabolism that catalyzes the reversible transfer of a phosphoryl group from MgATP to arginine, leading to phosphoarginine and MgADP [23]. Enzymes with two substrates often show synergism in substrate binding, that is, the binding of the first substrate facilitates the binding of the second substrate. The parameter Kd /Km is often used to represent synergism, where Kd is the dissociation constant in the absence of the second substrate [24]. Definition of it in mathematics is Kd /Km > 1; a higher ratio of Kd /Km indicates stronger synergism [24]. The substrate synergism may be associated with substrate induced conformational changes within the tertiary complex [25,6]. The synergism in substrate binding appears to be a common feature in phosphagen kinases [26]. Our study suggested that the substrate synergism has almost lost when the conserved V65 was replaced by the polar mutants. When V65 was replaced by nonpolar amino acids (V65G, V65A and V65L), the synergism in substrate binding
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also decreased, less than that of polar amino acids, Kd /Km value between 1.91 and 2.46. Analyzing the reason, the changes of synergism may be caused by polarity of the amino acids. When V65 was replaced by the polar amino acid (uncharged S or the negatively charged D), it led to significant changes in the substrate binding and synergism, which suggested that the amino acid V65 might play an important role in the substrate synergism. Protein folding is a process by which the amino acid sequence of a protein determines the three-dimensional conformation of the functional protein [27]. The proper folding of protein is important to maintain its function. Many amino acids located in the GS region not only play an important role in the guanidine recognition but also play important role in the AK structural stability and activity [28,29]. As seen from Figs. 3 and 4, the folding of the AKs was a complex process involving pre-molten globule (pre-MG) and molten globule (MG) intermediates. Although the position of the MG of mutant AK (0.3 M) was different from that of WT-AK (0.4 M), the similar value of the maximum ANS intensity suggested that mutant and WT AKs had similar populations of the pre-MG and MG state. The major difference between WT and the mutant AKs was the transition from the MG intermediate to the native state. It is seen that mutations altered the native ensembles of the enzyme, whose free energy was somewhat higher than that of native AK. For simplicity, the mutant could be taken as a state similar to the partially unfolded WT-AK denatured by low concentrations of denaturants. Although the refolding of WT-AK was not fully reversible in our in vitro conditions, the potential intracellular molecular chaperones and osmolytes might help WT-AK to achieve their native structure in vivo [30,31]. However, the mutant AK could not regain a functional state similar to that of the WT-AK through refolding, even if they could be trapped and assisted by chaperones. The aggregation rate of the mutants was much faster than that of WT-AK at all the three temperatures in this study. This result also suggested that the amino acid residue V65 mutation destabilized the structural stability of AK. Furthermore, the mutants were prone to aggregate when stored at room temperature for several days, while the WT-AK was not (data not shown). Combined with the spectroscopic results, one can deduce that the looser structure and more hydrophobic exposure induced by the amino acid residue V65 mutation might facilitate the protein to aggregate at elevated temperatures. The nature of all mutations existing in a partially folded state made it easier to be inactivated and unfolded upon environmental changes, and more prone to form insoluble aggregates. In conclusion, we investigated the role of V65 in AK substrate synergism, structural stability and activity. Our studies revealed that mutant in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism
alteration. Spectroscopic experiments indicated that the mutants were found to be in a partially folded state and more prone to aggregation when subjected to stresses. The results provided herein suggest that this conserved residue may play an important role in AK substrate synergism, structural stability and activity. Acknowledgements The present investigation was supported by Program for Changjiang Scholar and Innovative Research Team in University (IRT0635). References [1] T. Suzuki, Y. Kawasaki, T. Furukohri, Biochem. J. 328 (1997) 301–306. [2] W.R. Ellington, Ann. Rev. Physiol. 63 (2001) 289–325. [3] U. Schlattner, M. Tokarska-Schlattner, T. Wallimann, Biochim. Biophys. Acta 1762 (2005) 164–180. [4] M.J. McLeish, G.L. Kenyon, Crit. Rev. Biochem. Mol. Biol. 40 (2005) 1–20. [5] W.R. Ellington, T. Suzuki, in: C. Vial (Ed.), Molecular Anatomy and Physiology of Proteins-Creatine Kinase, Nova Science, New York, 2006, pp. 1–26. [6] G. Zhou, T. Somasundaram, E. Blanc, G. Parthsarthy, W.R. Ellington, M.S. Chapman, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 8449–8454. [7] M. Forstner, K. Manfred, P. Laggner, T. Wallimann, Biophys. J. 75 (1998) 1016–1023. [8] E.F. Pai, W. Sachsenheimer, R.H. Schirmer, G.E. Schulz, J. Mol. Biol. 114 (1997) 37–45. [9] G.H. Reed, M. Cohn, J. Biol. Chem. 247 (1972) 3073–3081. [10] K. Fritz-Wolf, T. Schnyder, T. Wallimann, W. Kabsch, Nature 381 (1996) 341–345. [11] T. Suzuki, Y. Kawasaki, T. Furukohri, W.R. Ellington, Biochim. Biophys. Acta 1348 (1997) 152–159. [12] T. Suzuki, M. Kamidochi, N. Inoue, H. Kawamichi, Y. Yazawa, T. Furukohri, R.W. Ellington, Biochem. J. 340 (1999) 671–675. [13] K. Uda, T. Suzuki, Protein J. 23 (2004) 53–64. [14] Q.Y. Wu, F. Li, X.Y. Wang, Insect. Biochem. Mol. Biol. 38 (2008) 59–65. [15] Q.Y. Wu, F. Li, X.Y. Wang, Int. J. Biol. Macromol. 43 (4) (2008) 367–372. [16] W.J. Zhu, M. Li, X.Y. Wang, Int. J. Biol. Macromol. 41 (2007) 564–571. [17] Q.Y. Wu, F. Li, W.J. Zhu, X.Y. Wang, Comp. Biochem. Phys. B 148 (2007) 355–362. [18] M. Li, X.Y. Wang, J.G. Bai, Protein Pept. Lett. 13 (2006) 405–410. [19] C.A. Pereira, G.D. Alonso, M.C. Paveto, J. Biol. Chem. 275 (2000) 1495–1501. [20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [21] K.K. Turoverov, S.Y. Haitlina, G.P. Pinaev, FEBS Lett. 62 (1976) 4–6. [22] N.A. Bushmarina, I.M. Kuznetsova, A.G. Biktashev, K.K. Turoverov, V.N. Uversky, Chembiochem 2 (2001) 819–821. [23] E.A. Newsholme, I. Beis, A.R. Leech, V.A. Zammit, Biochem. J. 172 (1978) 533–537. [24] W.W. Cleland, Enzymologia 63 (1979) 103–138. [25] E.T. Maggio, G.L. Kenyon, G.D. Markham, G.H. Reed, J. Biol. Chem. 252 (1977) 1202–1207. [26] S.D. Lahiri, P.F. Wang, P.C. Babbitt, M.J. McLeish, G.L. Kenyon, K.N. Allen, Biochemistry 41 (2002) 13861–13867. [27] C.B. Anfinisen, Science 181 (1973) 223–230. [28] Q. Guo, F. Zhao, S.Y. Guo, X.C. Wang, Biochimie 86 (2004) 379–386. [29] N. Fujimoto, K. Tanaka, T. Suzuki, FEBS Lett. 579 (2005) 1688–1692. [30] Y. Xia, Y.D. Park, H. Mu, H.M. Zhou, X.Y. Wang, F.G. Meng, Int. J. Biol. Macromol. 40 (2007) 437–443. [31] J.C. Pan, J.S. Wang, Y. Cheng, Z.H. Yu, X.M. Rao, H.M. Zhou, Biochem. Cell Biol. 83 (2005) 140–146.
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Val65 plays an important role in the substrate synergism, structural stability and activity of arginine kinase Qing-Yun Wu a,c,1 , Feng Li a,1 , Xiao-Yun Wang a,b,∗ a b c
College of Life Science, Shandong Agricultural University, Tai’an, Shandong 271018, People’s Republic of China State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, People’s Republic of China Institutes of Biochemistry and Cell Biology, State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 22 May 2009 Received in revised form 29 June 2009 Accepted 30 June 2009 Available online 21 July 2009 Keywords: Arginine kinase Mutagenesis Structural stability Substrate synergism Activity
a b s t r a c t Arginine kinase, a member of phosphagen kinase, is a key enzyme in the cellular energy metabolism of invertebrates. A series mutation of conserved amino acid residue V65 was constructed to investigate its role in AK substrate synergism, structural stability and activity. Our study revealed that mutation in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results provided herein suggest that amino acid residue V65 played a relatively important role in AK substrate synergism, structural stability and activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Arginine kinase (ATP: l-arginine phosphotransferase EC 2.7.3.3) (AK) is a member of what appears to be a highly conserved family of phosphotransferases that reversibly catalyze the transfer of phosphate from phosphoarginine to ADP, yielding ATP [1]. Both of these enzymes play an important role in cells, by buffering the ATP concentration according to cellular energy requirements [2–5]. Recently, a crystal structure for the transition state analog complex (TSAC) of the AK from the horseshoe crab Limulus has appeared [6]. Substrate binding in both CK and AK involves pronounced conformational changes [7–9], in which the two flexible loops move in such away as to clamp down on the substrates [7]. Although induced fit occurred in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be complicated [7–9]. The structure showed that four amino acid residues (Ser63, Gly64, Val65 and Tyr68 in Limulus AK sequence),
Abbreviations: AK, arginine kinase; CK, creatine kinase; IPTG, isopropyld-thiogalactopyranoside; ANS, 1-anilinonaphtalene-8-sulfonate; TSAC, transition state analog complex; GdnHCl, guanidine hydrochloride; Emax, emission maximum wavelength of the intrinsic fluorescence. ∗ Corresponding author at: College of Life Science, Shandong Agricultural University, Dai zong 61, Tai’an, Shandong 271018, People’s Republic of China. Tel.: +86 538 8242656 8430; fax: +86 538 8242217. E-mail address: [email protected] (X.-Y. Wang). 1 These authors contributed equally to this work. 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.06.016
located in the guanidine specificity region (GS region), might form hydrogen bond with the substrate arginine. This GS region was overlapped partly by the so-called flexible loop in the crystal structures of chicken mitochondrial CK [10] and Limulus AK [6]. There was a proportional relationship between the size of the deletion in the GS region and the mass of guanidine substrate used [11]. The amino acid residue Tyr68 was conserved in all AKs, except in the sea cucumber Stichopus AK that evolved from the CK gene, which suggested that the Tyr68 appeared to be the most important residue in substrate binding [12]. The amino acid residues Ser63, Gly64 were also reported to play essential roles in the substrate binding [13]. Alignment of amino acid sequences indicates that Val65 amino acid residue is conserved in all of guanidino kinases. Moreover, the structure analysis suggested that the amino acid residue Val65 might form a hydrogen bond with the amino group of the substrate arginine in Limulus AK, and we assumed that this residue might play a relative important role in substrate binding. However, little is known about the role of this amino acid residue Val65 in keeping AK structural stability, substrate synergism and activity. In our previous study, we demonstrated that some amino acid residues outside the active site played important roles in AK activity, substrate synergism and structural stability [14–16]. Does the amino acid residue V65 mutation affect AK function. In this study, the typical nonpolar amino acid valine, V65, was mutated to the non-typical nonpolar, uncharged and negatively charged amino acids of Gly (G), Ser (S) and Asp (D), respectively, to investigate its role in AK substrate synergism, structural stability and activity. The results suggested that this conserved residue may play an
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important role in AK substrate synergism, structural stability and activity. 2. Materials and methods 2.1. Cloning, site-directed mutagenesis and expression of the mutant locust AK pMD-18T plasmid with locust AK cDNA (pMD-Locust WT-AK) inserted was used as a template for mutagenesis [17]. The gene encodes 355 amino acids with a molecular weight of about 40 kDa. Five mutations (V65G, V65S, V65D, V65A and V65L) were introduced into the template of the pMD-locust WT-AK by overlap PCR using mutation primers. The sequences of these mutation primers for these mutations were as follows: for V65G, 5 CATGACTCTGGTGGTGGTATCTATGC-3 and 5 -GCATAGATACCACCACC AGAGTCATG-3 ; for V65S, 5 -ATGACTCTGGTTCTGGTATCTATGC-3 and 5 -GCATAGATACCAGAACCAGAGTCAT-3 ; for V65D, 5 CATGACTCTGGTGAC GGTATCTATGC-3 and 5 -GCATAGATACCGTCACCAGAGTCATG-3 , for V65A, 5 -CATGACTCTGGTGCTGGTATCTATGC-3 and 5 -GCATAGATACCAGCACCAGAG TCATG-3 , for V65L, 5 CATGACTCTGGTCTGGGTATCTATGC-3 and 5 -GCATA GATACCCAGACCAGA GTCATG-3 , mutated sequences were underlined. The PCR primers for the full-length gene were 5 -GATGGATCCATGGTTGATGCTGCAGTGCT G-3 and 5 -GACAAGCTTCAGAGTGCCTTACAGAGTCC-3 , in which the restriction sites introduced into the primers were underlined. Finally, the mutant products were completely sequenced to confirm that only the intended mutations had been introduced. The mutant gene was cloned into expression vector pET-28a and transformed into the Escherichia coli BL21 (DE3) codon plus. The His6 -tagged locust AK fusion protein was expressed in E. coli BL21 by induction with 2 mM isopropyl-1-thio--dgalactopyranoside (IPTG) at 25 ◦ C for 14 h. The soluble protein was extracted and purified using a nickel affinity chromatography column purification kit. The purity was checked by SDS-PAGE.
glycine–NaOH, 1 mM DTT at pH 8.6) for 24 h at equilibrium state. The refolding experiment was initiated by dilution of the denatured AKs into the standard buffer (pH 8.6) to final GdnHCl concentrations ranging from 0.1 M to 4 M at 25 ◦ C. The intrinsic and ANS fluorescence spectra of unfolding and refolding AKs were collected on an F-4500 spectrofluorometer using a 1-cm path-length cuvette being two Trp residues (position 214 and 221) in AK. 2.5. Parameter A and phase diagram analysis of intrinsic fluorescence data To more clearly characterize the effects of mutations on AK folding, Parameter A and phase diagram analysis, which were sensitive tools to identify folding intermediates, were used to compare the folding pathways of the WT and mutant AKs. Parameter A, which reflected the spectral shape of the intrinsic Trp fluorescence [21], was obtained by dividing the fluorescence intensity at 320 nm (I320 ) by the intensity at 365 nm (I365 ) during unfolding and refolding. The “phase diagram” analysis was carried out as described previously [22]. The fluorescence data were normalized by I320 and I365 of the spectra during unfolding and refolding. In the phase diagram, the joint position of two lines indicated that an intermediate appeared at the corresponding GdnHCl concentration. 3. Results 3.1. Expression of the mutant locust AK and enzymatic analysis All of the recombinant enzymes were successfully expressed as soluble fusion proteins with His6 -tag, and purified by affinity chromatography and size exclusion chromatography. The enzymatic characteristics of the recombinant wild-type (WT) fusion enzymes were very similar to that of native locust AK, indicating the Nterminal His6 -tag portion had no effect on their activity. 3.2. Kinetic parameters analysis
2.2. Enzyme assay and determination of kinetic parameters AK activity (phosphoarginine synthesis) was assayed using a modification of previous procedures [18,19]. The assay mixture for AK activity determination consisted of 100 mM Tris (pH 8.0), 10 mM l-arginine, 2 mM ATP-Na, 10 mM mercaptoethanol and 10 L of 0.01 mM enzyme solution. The absorbance at 660 nm was measured at 30 ◦ C using an Ultrospec4300 pro UV-vis Spectrophotometer. Protein concentration was determined by Bradford’ method [20], using an Ultrospec4300 pro UV-vis Spectrophotometer. The two-substrate graphical method was used to obtain the kinetic parameters [14]. The enzyme assays were carried out at the optimum pH (pH 8.6) and temperature (30 ◦ C) with different concentrations of ATP and arginine. And all these reactions were carried out at least four times. 2.3. Thermal stability of AK The thermal stability of WT and mutants AKs was determined by activity assay after incubation at different temperatures. The enzyme solutions were incubated at given temperatures varying from 25 ◦ C to 65 ◦ C for 10 min, then cooled on ice and the activity was measured at 30 ◦ C. The aggregation of AKs at given temperature was monitored by measuring the turbidity at 400 nm. 2.4. Unfolding and refolding experiments The native and mutant AKs were added to different concentrations of GdnHCl dissolved in the standard buffer (10 mM
Table 1 shows the effects of mutations of V65 on AK enzyme kinetics. The catalytic constants of mutations decreased in different degrees, about 86.7%, 73.5%, 62.6%, 57.3% and 33.4% of V65G, V65A, V65L, V65S and V65D compared with that of WT-AK, respectively. It shows when the typical nonpolar amino acid V65 was replaced by polar amino acids (V65D and V65S), the enzyme activities decreased most. Similarly, the binding affinity of arginine and ATP in the mutant AKs of polar amino acids was lower than nonpolar mutations, compared to that of WT-AK, as indicated by about 2- to 3-folds increase of the Km values for ATP and arginine. However, the Kd values for arginine and ATP of these mutants were increased a little, about 1.14- to 1.37-folds compared with that of WT-AK, resulting in a Kd /Km value of 1.08–1.56, indicating that the synergism in substrate binding of WT-AK (Kd /Km value 2.80) was decreased. More importantly, the substrate synergism for the mutation V65D Arg had almost lost and its kcat /Km value (18.94 s−1 mM−1 ) showed 9-folds decline in enzyme catalysis efficiency, compared with that (167.6 s−1 mM−1 ) of WT-AK. When V65 was replaced by nonpolar amino acids (V65G, V65A and V65L), the synergism in substrate binding also decreased, less than that of polar amino acids, Kd /Km value between 1.91 and 2.46. The changes of synergism may be caused by polarity of the amino acids. To sum up, when V65 was replaced by the nonpolar amino acids it led to moderate changes, while when it was replaced by the polar amino acid (uncharged S or the negatively charged D), it led to significant changes in the substrate binding and synergism. Glycine is special in polarity, between nonpolar and polar amino acids. The replacement of the residues in the mutants (D and S) might lead to the much looser tertiary
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Table 1 Comparison of kinetic parameters for the forward reaction of recombinant WT and mutant AKs. Vmax , mol Pi min−1 mg−1
Mutants a
Recombinant WT AK V65G V65S V65D V65A V65L
239.1 208.5 136.8 78.2 176.3 149.9
± ± ± ± ± ±
7.65 3.61 3.11 3.04 4.04 3.66
Arg
kcat , s−1 159.4 138.4 91.3 53.2 117.2 99.8
± ± ± ± ± ±
Km , mM 6.2 1.81 2.11 1.91 1.82 1.69
0.951 1.48 2.15 2.81 1.69 1.85
± ± ± ± ± ±
0.08 0.15 0.23 0.31 0.21 0.19
Arg
Kd , mM 2.67 3.64 3.35 3.04 3.62 3.53
± ± ± ± ± ±
0.22 1.05 0.72 0.51 0.99 1.08
ATP Km , mM
1.270 2.028 3.11 3.71 2.26 2.47
± ± ± ± ± ±
0.23 0.52 0.44 0.75 1.05 1.02
Arg
Kd
,
3.56 5.13 4.85 4.01 4.93 4.72
Arg
mM
kcat /Km , s−1 mM
± ± ± ± ± ±
167.6 93.51 42.5 18.9 69.4 53.9
0.32 1.84 1.13 0.54 1.77 1.66
± ± ± ± ± ±
Kd /Km
5.34 4.36 3.31 0.89 2.98 2.53
2.80 2.46 1.56 1.08 2.18 1.91
± ± ± ± ± ±
0.66 0.99 0.24 0.36 0.78 0.64
Note: Kinetic parameters were obtained from at least three runs of the reaction. a Kinetic parameters cited from the article [14].
structures than that of WT-AK. Now that this amino acid did not participate in the catalytic mechanism of AK and the substrate binding of ATP, the effects of the mutation on the substrate binding affinity of ATP and the substrate synergism of AK might be due to the impaired tertiary structure of AK. 3.3. Effects of mutations on AK structure Concerning the tertiary structure, the emission maximum of the intrinsic fluorescence (Fig. 1A) was found to red shift to about 333 nm for V65G, about 336 nm for V65S and about 338 nm for V65D compared to the WT-AK (331 nm), which suggested that the Trp residues in mutant AKs were more exposed to water and the tertiary structures of the mutations were much looser than that of WT-AK. The fluorescence spectrum of V65A and V65L were almost identical with WT-AK (data not shown). The ANS fluorescence intensity of all mutations was higher than that of WT-AK at the same concentration (Fig. 1B) which reflected that the mutant AKs had more hydrophobic exposure than that of WT-AK to allow the binding of the ANS molecules. These spectroscopic experiments clearly indicated that the mutations impaired the tertiary structures of AK. Analyzing the reason, the replacement of the hydrophobic by hydrophilic amino acid would weaken the hydrophobic interactions which may essential in sustaining AK structure stability and finally result in the more hydrophobic exposure and the much looser tertiary structures. 3.4. Effects of mutations on AK thermal stability and thermal aggregation The effects of mutations on AK stability were evaluated by the residual activity retained after 10 min heat treatment (Fig. 2A). The activity of WT-AK had few changes after heat treatment at temperatures below 40 ◦ C. A steep decrease of activity was observed
between 40 ◦ C and 60 ◦ C, and a complete loss in activity occurred above 65 ◦ C. All the mutations were much less stable than that of WT-AK. The midpoints of thermal inactivation of WT-AK and the mutations V65G, V65A, V65L, V65S and V65D were about 53.6 ± 0.5 ◦ C, 49.4 ± 0.5 ◦ C, 50.2 ± 0.5 ◦ C, 48.3 ± 0.5 ◦ C, 43.6 ± 0.5 ◦ C and 39.3 ± 0.5 ◦ C, respectively. The aggregation experiment also showed the similar effects of mutations on AK conformation. Only a little aggregation occurred after heated at 41 ◦ C for 60 min for WT-AK, whereas considerable aggregation was observed for all mutations at 45 ◦ C (Fig. 2B). When the heating temperature was increased to 54 ◦ C, the aggregation rate of mutations was much faster than that of WT-AK. Combining the above results, we can deduce that in the mutations the impaired structure and greater hydrophobic exposure might facilitate protein aggregation at elevated temperatures because the hydrophobic amino acid was replaced by the hydrophilic amino acid. 3.5. Effects of the mutations on AK unfolding and refolding Spectroscopic methods were used to investigate the effects of mutations on AK unfolding and refolding. As shown in Fig. 3A and B, in the unfolding and refolding process, increasing or decreasing GdnHCl concentrations caused a sharp red or blue shift of the emission maximum. However, in the ranges of 0.2–0.5 M and 0.9–1.2 M GdnHCl, there was little change in the emission maximum for the mutant, while that of WT-AK was 0.3–0.6 M and 0.9–1.2 M GdnHCl. Meanwhile, the ANS intensity reached a maximum at 0.3 M and 1.0 M GdnHCl for the mutations, while that of the WT-AK was at 0.4 M and 1.0 M GdnHCl, as was shown in Fig. 3C and D. (The results of V65A and V65L were almost identical with that of V65G, so only V65G was shown here.) These results implied that two equilibrium intermediate states existed in the folding process of WT and mutant AKs although there was little different in concentration
Fig. 1. Effects of mutations on AK tertiary structures, intrinsic fluorescence spectra (A), and ANS fluorescence spectra (B). Curve (1) represents spectra of WT-AK, (2) V65G, (3) V65S, (4) V65D, and (5) represents the emission fluorescence spectra of ANS solution. The WT and mutant AK was dissolved in standard buffer at a final concentration of 3.2 M. ANS fluorescence spectra were recorded using an excitation wavelength of 380 nm.
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Fig. 2. Thermal stability and aggregation of AKs. (A) Thermal stability of AKs. (B) Thermal aggregation of AKs at 45 ◦ C.
of GdnHCl that the equilibrium intermediate states formation. The first intermediate was very similar to that of the pre-molten globule state with no activity, and the second has the characteristics of the highly ordered molten globule state with partial activity, which was quite consistent with previous observations [22,23]. The transitions of WT and mutant AKs refolding from the GdnHCl-denatured state were similar to those unfolding at GdnHCl concentrations above 0.5 M when monitored by these two techniques. The deviation between the unfolding and refolding at GdnHCl concentrations below 0.5 M indicated that the folding of WT and mutant AKs was not fully reversible under our conditions. Nevertheless, the transition curves of the mutant AKs were almost identical to that of WT-AK at GdnHCl concentrations above 0.5 M, which suggested that the mutation did not affect the M↔G↔U transitions. The major
difference in folding between WT-AK and mutations was observed at low GdnHCl concentrations. All these results suggested that the major differences in folding between WT-AK and mutations at low GdnHCl concentrations might be caused by the differences in their native structures (Fig. 1). Parameter A and phase diagram analysis were also used to clarify the effects of mutations on AK folding. As presented in Fig. 4A and B, the unfolding transition curves of mutations V65S, V65D and V65G were almost identical to WT-AK at all conditions for GdnHCl concentrations varying from 0.5 M to 6 M. Furthermore, as indicated by the phase diagram analysis in Fig. 4C and D, all the transitions could be fitted by three linear parts, which suggested that all the transitions were a three independent transitions process with the appearance of two intermediates although the GdnHCl concentra-
Fig. 3. Unfolding and refolding of WT and mutant AKs, maximum emission wavelength (Emax ) of the intrinsic fluorescence (A) and the intensity of ANS fluorescence (C) in unfolding, maximum emission wavelength (Emax ) of the intrinsic fluorescence (B) and the intensity of ANS fluorescence (D) in refolding. The final concentration of AK was 3.2 M.
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Fig. 4. Parameter A (A–C) and phase diagram (D–F) analysis of intrinsic fluorescence data in unfolding. Parameter A was obtained by dividing the fluorescence intensity at 320 nm (I320 ) by the intensity at 365 nm (I365 ). The phase diagram was constructed by I320 vs. I365 for the unfolding of AK.
tion of the appearance of the first intermediates of the mutant (0.3 M) was different from that of WT-AK (0.4 M). The joint position of the two lines above 0.5 M was also at the same GdnHCl concentration, when below the 0.5 M the joint position of the two lines was at 0.3 M for the mutant, while that of the WT-AK was 0.4 M. In the refolding experiments, the refolding transition curves of all the mutations showed the same results (data not shown). These results coincided with those from direct spectra analysis in Fig. 3, which also suggested that the mutation did not affect the folding pathways of AK. The discrepancy between the WT and the mutant unfolding at low concentrations of GdnHCl was more likely to be caused by their different native structures, but not by the interference of the folding pathways by the mutation. 4. Discussion Phosphagen (guanidino) kinases contain a family of highly conserved enzymes, which catalyze the reversible transfer of phos-
phate from phosphagen such as creatine phosphate to ADP. AK similar to CK in vertebrates is a phosphagen kinase participating in cell metabolism that catalyzes the reversible transfer of a phosphoryl group from MgATP to arginine, leading to phosphoarginine and MgADP [23]. Enzymes with two substrates often show synergism in substrate binding, that is, the binding of the first substrate facilitates the binding of the second substrate. The parameter Kd /Km is often used to represent synergism, where Kd is the dissociation constant in the absence of the second substrate [24]. Definition of it in mathematics is Kd /Km > 1; a higher ratio of Kd /Km indicates stronger synergism [24]. The substrate synergism may be associated with substrate induced conformational changes within the tertiary complex [25,6]. The synergism in substrate binding appears to be a common feature in phosphagen kinases [26]. Our study suggested that the substrate synergism has almost lost when the conserved V65 was replaced by the polar mutants. When V65 was replaced by nonpolar amino acids (V65G, V65A and V65L), the synergism in substrate binding
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also decreased, less than that of polar amino acids, Kd /Km value between 1.91 and 2.46. Analyzing the reason, the changes of synergism may be caused by polarity of the amino acids. When V65 was replaced by the polar amino acid (uncharged S or the negatively charged D), it led to significant changes in the substrate binding and synergism, which suggested that the amino acid V65 might play an important role in the substrate synergism. Protein folding is a process by which the amino acid sequence of a protein determines the three-dimensional conformation of the functional protein [27]. The proper folding of protein is important to maintain its function. Many amino acids located in the GS region not only play an important role in the guanidine recognition but also play important role in the AK structural stability and activity [28,29]. As seen from Figs. 3 and 4, the folding of the AKs was a complex process involving pre-molten globule (pre-MG) and molten globule (MG) intermediates. Although the position of the MG of mutant AK (0.3 M) was different from that of WT-AK (0.4 M), the similar value of the maximum ANS intensity suggested that mutant and WT AKs had similar populations of the pre-MG and MG state. The major difference between WT and the mutant AKs was the transition from the MG intermediate to the native state. It is seen that mutations altered the native ensembles of the enzyme, whose free energy was somewhat higher than that of native AK. For simplicity, the mutant could be taken as a state similar to the partially unfolded WT-AK denatured by low concentrations of denaturants. Although the refolding of WT-AK was not fully reversible in our in vitro conditions, the potential intracellular molecular chaperones and osmolytes might help WT-AK to achieve their native structure in vivo [30,31]. However, the mutant AK could not regain a functional state similar to that of the WT-AK through refolding, even if they could be trapped and assisted by chaperones. The aggregation rate of the mutants was much faster than that of WT-AK at all the three temperatures in this study. This result also suggested that the amino acid residue V65 mutation destabilized the structural stability of AK. Furthermore, the mutants were prone to aggregate when stored at room temperature for several days, while the WT-AK was not (data not shown). Combined with the spectroscopic results, one can deduce that the looser structure and more hydrophobic exposure induced by the amino acid residue V65 mutation might facilitate the protein to aggregate at elevated temperatures. The nature of all mutations existing in a partially folded state made it easier to be inactivated and unfolded upon environmental changes, and more prone to form insoluble aggregates. In conclusion, we investigated the role of V65 in AK substrate synergism, structural stability and activity. Our studies revealed that mutant in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism
alteration. Spectroscopic experiments indicated that the mutants were found to be in a partially folded state and more prone to aggregation when subjected to stresses. The results provided herein suggest that this conserved residue may play an important role in AK substrate synergism, structural stability and activity. Acknowledgements The present investigation was supported by Program for Changjiang Scholar and Innovative Research Team in University (IRT0635). References [1] T. Suzuki, Y. Kawasaki, T. Furukohri, Biochem. J. 328 (1997) 301–306. [2] W.R. Ellington, Ann. Rev. Physiol. 63 (2001) 289–325. [3] U. Schlattner, M. Tokarska-Schlattner, T. Wallimann, Biochim. Biophys. Acta 1762 (2005) 164–180. [4] M.J. McLeish, G.L. Kenyon, Crit. Rev. Biochem. Mol. Biol. 40 (2005) 1–20. [5] W.R. Ellington, T. Suzuki, in: C. Vial (Ed.), Molecular Anatomy and Physiology of Proteins-Creatine Kinase, Nova Science, New York, 2006, pp. 1–26. [6] G. Zhou, T. Somasundaram, E. Blanc, G. Parthsarthy, W.R. Ellington, M.S. Chapman, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 8449–8454. [7] M. Forstner, K. Manfred, P. Laggner, T. Wallimann, Biophys. J. 75 (1998) 1016–1023. [8] E.F. Pai, W. Sachsenheimer, R.H. Schirmer, G.E. Schulz, J. Mol. Biol. 114 (1997) 37–45. [9] G.H. Reed, M. Cohn, J. Biol. Chem. 247 (1972) 3073–3081. [10] K. Fritz-Wolf, T. Schnyder, T. Wallimann, W. Kabsch, Nature 381 (1996) 341–345. [11] T. Suzuki, Y. Kawasaki, T. Furukohri, W.R. Ellington, Biochim. Biophys. Acta 1348 (1997) 152–159. [12] T. Suzuki, M. Kamidochi, N. Inoue, H. Kawamichi, Y. Yazawa, T. Furukohri, R.W. Ellington, Biochem. J. 340 (1999) 671–675. [13] K. Uda, T. Suzuki, Protein J. 23 (2004) 53–64. [14] Q.Y. Wu, F. Li, X.Y. Wang, Insect. Biochem. Mol. Biol. 38 (2008) 59–65. [15] Q.Y. Wu, F. Li, X.Y. Wang, Int. J. Biol. Macromol. 43 (4) (2008) 367–372. [16] W.J. Zhu, M. Li, X.Y. Wang, Int. J. Biol. Macromol. 41 (2007) 564–571. [17] Q.Y. Wu, F. Li, W.J. Zhu, X.Y. Wang, Comp. Biochem. Phys. B 148 (2007) 355–362. [18] M. Li, X.Y. Wang, J.G. Bai, Protein Pept. Lett. 13 (2006) 405–410. [19] C.A. Pereira, G.D. Alonso, M.C. Paveto, J. Biol. Chem. 275 (2000) 1495–1501. [20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [21] K.K. Turoverov, S.Y. Haitlina, G.P. Pinaev, FEBS Lett. 62 (1976) 4–6. [22] N.A. Bushmarina, I.M. Kuznetsova, A.G. Biktashev, K.K. Turoverov, V.N. Uversky, Chembiochem 2 (2001) 819–821. [23] E.A. Newsholme, I. Beis, A.R. Leech, V.A. Zammit, Biochem. J. 172 (1978) 533–537. [24] W.W. Cleland, Enzymologia 63 (1979) 103–138. [25] E.T. Maggio, G.L. Kenyon, G.D. Markham, G.H. Reed, J. Biol. Chem. 252 (1977) 1202–1207. [26] S.D. Lahiri, P.F. Wang, P.C. Babbitt, M.J. McLeish, G.L. Kenyon, K.N. Allen, Biochemistry 41 (2002) 13861–13867. [27] C.B. Anfinisen, Science 181 (1973) 223–230. [28] Q. Guo, F. Zhao, S.Y. Guo, X.C. Wang, Biochimie 86 (2004) 379–386. [29] N. Fujimoto, K. Tanaka, T. Suzuki, FEBS Lett. 579 (2005) 1688–1692. [30] Y. Xia, Y.D. Park, H. Mu, H.M. Zhou, X.Y. Wang, F.G. Meng, Int. J. Biol. Macromol. 40 (2007) 437–443. [31] J.C. Pan, J.S. Wang, Y. Cheng, Z.H. Yu, X.M. Rao, H.M. Zhou, Biochem. Cell Biol. 83 (2005) 140–146.