1 Department of Applied Biological Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, 2 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 and 3 Department of Bioscience and Biotechnology, Aomori University, Aomori 030-0943, Japan
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Abstract |
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Keywords: allosteric enzyme/fructose 1,6-bisphosphate/L-lactate dehydrogenase/lactic acid bacteria/Lactobacillus casei
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Introduction |
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Among known L-LDHs, Lactobacillus casei L-LDH shows unique allosteric properties (Holland and Pritchard, 1975; Hensel et al., 1977
, 1983
; Garvie, 1980
). Whereas the enzyme absolutely requires Fru(1,6)P2 for its catalytic activity under neutral conditions, it exhibits marked catalytic activity even in the absence of Fru(1,6)P2 under acidic conditions, where the enzyme gives sigmoidal and hyperbolic saturation curves for substrate pyruvate without and with Fru(1,6)P2, respectively. Kinetic studies (Gordon and Doelle, 1976
; Hensel et al., 1983
) and affinity labeling with 3-bromopyruvate (Hensel et al., 1983
) have revealed that pyruvate is bound to an allosteric binding site, which corresponds to the Fru(1,6)P2-binding site, when Fru(1,6)P2 is absent.
Unlike the L.casei enzyme, the L-LDH of Lactobacillus pentosus, previously called Lactobacillus plantarum, is a non-allosteric enzyme, like the vertebrate enzymes (Taguchi and Ohta, 1992; Arai et al., 2001
; Uchikoba et al., 2002
), although the two Lactobacillus enzymes show particularly high amino acid sequence identity (Taguchi and Ohta, 1991
, 1992
; Griffin et al., 1992
). Among the 10 residues of the Fru(1,6)P2-binding site, the L.casei and L.pentosus enzymes only differ in the amino acid residues at positions 188 and 205 (residues are numbered according to Eventoff et al., 1977
), the former enzyme possessing His residues, but the latter Asp and Thr, respectively (Taguchi and Ohta, 1992
). The site-specific replacement of His188 with Asp completely abolishes the heterotropic effect of Fru(1,6)P2 on the L.casei enzyme (Taguchi and Ohta, 1995
), indicating that His188 is essential for the regulation by Fru(1,6)P2, as in the cases of the well studied Fru(1,6)P2-regulated L-LDHs (Schröder et al., 1988
; Clarke et al., 1989
; Iwata et al., 1994
). In this work, we describe the replacement of His205 with Thr, which did not induce a marked reduction in the enzyme activation by Fru(1,6)P2, but a great loss of the Fru(1,6)P2-independent activation of the enzyme by a substrate, in contrast with the case of His188 replacement.
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Materials and methods |
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Oligodeoxynucleotide 5'-GAT GTT AGC AGT AGC TTG CCA TAC AGG-3' was purchased from Takara Syuzo and used to replace His205 with Thr. Site-directed mutagenesis was performed with a MUTA-GENE in vitro mutagenesis kit (Bio-RAD) according to Kunkel (Kunkel, 1985). Cultivation of recombinant Escherichia coli cells harboring expression plasmids for the gene encoding L.casei IAM 12473 (=ATCC 393) wild-type L-LDH, and H188D and H205T mutant L-LDHs, in which His188 and His205 are replaced with Asp and Thr, respectively, and purification of these three types of enzyme were performed essentially as described previously (Taguchi and Ohta, 1995
). The purity of the enzyme preparations was examined by SDSPAGE according to Laemmli (Laemmli, 1970
). The enzyme samples for chemical modification with 3-bromopyruvate were confirmed to be coenzyme-free by measurement of the absorbance at 260 and 280 nm before use.
Enzyme assay and protein determination
The assaying of the purified enzymes was carried out at 30°C in 50 mM sodium acetate buffer (pH 5.0) or 50 mM sodium morpholinepropanesulfonic acid (MOPS) buffer (pH 7.0) containing 0.1 mM NADH. One unit was defined as the catalytic rate of the conversion of 1 µmole of substrate per minute. Protein concentrations were determined with Bio-RAD Protein Assay protein reagent (Bio-RAD), using bovine serum albumin as a standard protein. Since the catalytic reaction with the L.casei enzyme in the presence of Fru(1,6)P2 was markedly inhibited by high concentrations of substrates, kinetic parameters such as KM and maximal velocity (Vmax) were estimated using only data obtained with substrate concentrations sufficiently low and non-inhibitory to give linear lines on double-reciprocal plotting. For the reactions that showed significant cooperativity, the Hill equation (Dixon and Webb, 1979) was used for sigmoidal curve fitting to obtain kinetic parameters such as the Hill coefficient (h) and half-saturating substrate concentration (S0.5). Inhibition constants for phosphoenolpyruvate, inorganic phosphate and 2-ketoglutarate were determined essentially according to a previous report (Hensel et al., 1983
).
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Results |
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Unlike the wild-type L.casei L-LDH, the H205T mutant enzyme, in which His205 is replaced with Thr, exhibited no marked catalytic activity in the absence of Fru(1,6)P2 at pH 5.0, even when the pyruvate concentration was increased to 50 mM (Figure 1). Nevertheless, the reaction with the H205T enzyme was greatly enhanced in the presence of 5 mM Fru(1,6)P2, the shape of the saturation curve being hyperbolic for pyruvate, as in the case of the wild-type enzyme. The kinetic parameters of the enzymes for pyruvate saturation are summarized in Table I
, together with those of the wild-type enzyme and the H188D enzyme, in which His188 is replaced with Asp. In the presence of 5 mM Fru(1,6)P2, the H205T enzyme exhibited a slightly increased KM value and virtually the same Vmax value compared to the wild-type enzyme, whereas the activation effect of Fru(1,6)P2 was lost in the H188D enzyme.
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It is known that pyruvate analogs instead of Fru(1,6)P2 also stimulate the reaction with L.casei L-LDH under acidic conditions (Hensel et al., 1983). In the case of the wild-type or H188D enzyme, two pyruvate analogs, oxamate and 2-ketoglutarate, apparently stimulated the catalytic activity by reducing the homotropic cooperativity, and the latter also induced a marked increase in the Vmax value, beside significant reduction of the S0.5 and h values (Figure 1
and Table I
). In contrast to these two types of enzymes, the H205T enzyme exhibited too low activity in the presence of 10 mM oxamate or 2-ketoglutarate to determine the exact kinetic parameters. Figure 2
shows the activities of the H205T enzyme at pH 5.0 in the presence of various concentrations of oxamate and 2-ketoglutarate with a fixed concentration of pyruvate (1 mM), together with the activity of the wild-type enzyme. Oxamate exhibited only a slight activation effect on the H205T mutant enzyme compared to the wild-type enzyme, and 2-ketoglutarate had no marked effect on the H205T enzyme up to 50 mM, in contrast to the case of the wild-type enzyme.
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It is known that L.casei L-LDH absolutely requires Fru(1,6)P2 for its enzyme activity under neutral conditions, where the activation function of Fru(1,6)P2 is markedly enhanced in the presence of certain divalent metal ions such as Mn2+ (Holland and Pritchard, 1975; Gordon and Doelle, 1976
; Hensel et al., 1977
). As in the case of the wild-type enzyme, the H205T enzyme exhibited no detectable catalytic activity at pH 7.0 in the absence of Fru(1,6)P2, but significant activity in the presence of 5 mM Fru(1,6)P2, the pyruvate saturation curve being sigmoidal (Figure 3
). In addition, MnSO4 (10 mM) also stimulated the catalytic reaction with the H205T enzyme in the presence of 5 mM Fru(1,6)P2, the sigmoidal pyruvate saturation curve changing into a hyperbolic one. The kinetic parameters of these enzymes at pH 7.0 are also shown in Table I
. The H205T enzyme exhibited essentially the same catalytic properties as the wild-type enzyme, and similar kinetic parameters, i.e. S0.5, h and Vmax values, to those of the wild-type enzyme, unlike in the case of pH 5.0.
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The activation function of Fru(1,6)P2 as to the H205T enzyme was evaluated at pH 5.0 and 7.0 in the presence of 1.0 and 10 mM pyruvate, respectively (Figure 4 and Table II
). At pH 5.0, the wild-type L.casei enzyme was much more (~2.5x 104-fold) sensitive to Fru(1,6)P2 than at pH 7.0, the S0.5 for Fru(1,6)P2 being 2 µM (Table II
). The H205T enzyme also exhibited high sensitivity to Fru(1,6)P2 at pH 5.0, but required a slightly higher (4-fold) concentration of Fru(1,6)P2 for activation than the wild-type enzyme. Like the wild-type enzyme, the H205T enzyme also required a much higher concentration of Fru(1,6)P2 for the activity at pH 7.0, when MnSO4 was absent. In this case, however, the H205T enzyme exhibited slightly higher sensitivity (a 2.5-fold lower S0.5 value) to Fru(1,6)P2 than the wild-type enzyme, unlike in case of pH 5.0. Consequently, the H205T enzyme exhibited only a 2.5x103-fold lower (10-fold different from the case of the wild-type enzyme) Fru(1,6)P2 sensitivity at pH 7.0 than at pH 5.0. As in the case of the wild-type enzyme, the activation function of Fru(1,6)P2 at pH 7.0 with the H205T enzyme was markedly improved by 10 mM MnSO4.
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It is known that 3-bromopyruvate specifically modifies His188 of L.casei L-LDH in the absence of the coenzyme NADH, and thereby leads to desensitization of the enzyme to Fru(1,6)P2, indicating that pyruvate or an analog binds to the allosteric site (Hensel et al., 1983). Therefore, to evaluate the effect of the replacement of His205 on the pyruvate binding to the allosteric site, the wild-type and H205 mutant enzymes were treated with 10 mM 3-bromopyruvate with and without 10 mM oxamate (Figure 5
). As in the case of the wild-type enzyme, 3-bromopyruvate also induced marked desensitization of the H205T enzyme to Fru(1,6)P2. The decrease in the Fru(1,6)P2-sensitive enzyme indicated an apparent first-order reaction, and gave the same half-life of 2.1 h for the wild-type and H205T enzymes in the absence of oxamate. In the presence of 10 mM oxamate, the desensitization was significantly inhibited in the cases of both the wild-type and H205T enzymes, although the latter enzyme had a slightly shorter half-life (2.7 h) than that of the former enzyme (3.3 h).
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It has been reported that phosphoenolpyruvate and 2-ketoglutarate competitively inhibit the activation function of Fru(1,6)P2 as to the wild-type L.casei L-LDH under acidic conditions, whereas they exhibit weak but significant activation effects in the absence of Fru(1,6)P2 (Hensel et al., 1983). As in the case of the wild-type enzyme, both phosphoenolpyruvate and 2-ketoglutarate exhibited marked inhibitory effects on the H205T enzyme through competition with Fru(1,6)P2, and gave only slightly increased (2.4- to 2.3-fold, respectively) inhibition constants (KI) as compared with the case of the wild-type enzyme (Table III
); however, they exhibited virtually no activation effects on the H205T enzyme in the absence of Fru(1,6)P2, unlike in the case of the wild-type enzyme. On the other hand, as compared with these two inhibitors, inorganic phosphate is a more general inhibitor competing with the activation by Fru(1,6)P2 of many Fru(1,6)P2-dependent L-LDHs (Garvie, 1980
) including the L.casei enzyme (Gordon and Doelle, 1976
). Inorganic phosphate also exhibited an inhibitory effect on the H205T enzyme as in the case of the wild-type enzyme, but gave a slightly less increased KI as compared with phosphoenolpyruvate and 2-ketoglutarate (Table III
).
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Table IV shows a comparison of the 10 amino acids comprising the Fru(1,6)P2-binding site, or the corresponding site in the case of non-allosteric enzymes, among L-LDHs of L.casei-related lactic acid bacteria and other representative L-LDHs. As compared with Arg173 and His188, which are essential for the regulation by Fru(1,6)P2 of general Fru(1,6)P2-activated L-LDHs (Clarke et al., 1987
, 1989
; Matsuzawa et al., 1988
; Schröder et al., 1988
), His205 is poorly conserved in known allosteric and non-allosteric L-LDHs, and is actually missing in the well studied enzymes such as the B.stearothermophilus, T.caldophilus and B.longum enzymes, although the latter two are activated through the cooperative effects of substrates in the absence of Fru(1,6)P2 (Taguchi et al., 1984
; Matsuzawa et al., 1988
; Fushinobu et al., 1996
, 1998
), as in the case of the L.casei L-LDH. On the other hand, the amino acids of the allosteric site are well conserved in the enzymes of some lactic acid bacteria related to L.casei, as compared with the case of other L-LDHs (Table IV
). As well as His188, His205 is conserved in the Fru(1,6)P2-dependent enzymes of Lactococcus lactis, Streptococcus mutans and Strepococcus thermophilus enzyme, which are Fru(1,6)P2-dependent L-LDHs except for the last one (Ito and Sasaki, 1994
). In contrast, the L.pentosus (Taguchi and Ohta, 1992
), L.plantarum (Ferain et al., 1994
) and Pediococcus acidilactici (Garmyn et al., 1995
) L-LDHs, which constitute a group of non-allosteric L-LDHs, specifically lack both His205 and His188.
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Discussion |
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Thus, His205 replacement did not markedly impair the heterotropic effect of Fru(1,6)P2 or Mn2+, but greatly diminished the activating effects of substrates (analogs) in the absence of Fru(1,6)P2, leading to an absolute Fru(1,6)P2 requirement for the enzyme activity of the L.casei L-LDH, in contrast to the case of the replacement of His188. This suggests that only a small structural change such as a single amino acid replacement may sufficiently change such absolute or non-absolute Fru(1,6)P2 dependence of allosteric L-LDHs.
In the case of the B.longum alosteric L-LDH, it has been reported that the allosteric transition can be assumed as two states, the inactive (T) and active (R) states, according to the concerted model demonstrated by Monod et al. (Monod et al., 1965), and the allosteric and catalytic sites communicate with each other through the quaternary structure change (Iwata et al., 1994
; Fushinobu et al., 1996
, 1998
). In the concerted model, a homotropic regulation for allosteric L-LDHs is described by the equation:
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The three-dimensional structures of the T and R states of the B.longum enzyme (Iwata et al., 1994) imply that one of the potent triggers for the Fru(1,6)P2-induced enzyme activation is the strong ionic interaction between the Arg173 guanidino group and the Fru(1,6)P2 phosphate moiety, in which the motion of Arg173 to the Fru(1,6)P2 phosphate coordinates the allosteric change of the enzyme conformation with the R state. Figure 6
shows the allosteric sites of the T and R states of the B.longum L-LDH (A), together with that of the Fru(1,6)P2-bound B.stearothermophilus enzyme (Wigley et al., 1992
) and the corresponding site for the non-allosteric L.pentosus (Uchikoba et al., 2001) enzyme (B), both of which can be considered to be in the R state. As in the case of Arg173, the amino acid at position 205 of the B.longum enzyme (Ser205) also significantly moves across the P-axis dimer interface during the allosteric transition of the B.longum enzyme, suggesting that this residue may also potentially trigger the allosteric transition through the formation of some interaction networks. Since the Fru(1,6)P2-independent activation by a substrate highly depends on acidic conditions, only the protonated form of His205 is possibly able to participate in such interaction networks.
Although one of the possible ligands for His205 is a pyruvate molecule bound to the allosteric site, it is not evident as to whether or not His205 directly interacts with pyruvate in the allosteric site. The H205T enzyme was desensitized to Fru(1,6)P2 by 3-bromopyruvate, and protected from the desensitization by oxamate in a similar way to the case of the wild-type enzyme (Figure 5). In addition, the mutant enzyme exhibited only slightly increased KI values for pyruvate analogs that compete with Fru(1,6)P2 (Table III
). These results indicate that His205 is not essential for the substrate binding to the allosteric site, at least when the enzyme is in the T state conformation, which should have been predominant in these experiments.
Another possible ligand for His205 is some amino acid residue(s) of the protein, possibly on the P-axis interface of the counterpart subunit related through the P-axis, toward which His205 appears to move during the conformational change to the R state (Figure 6). Using X-ray crystallography of several types of L.casei L-LDH, we have found that the imidazole of His205 can form an ionic hydrogen bond with amino acids, such as Glu211, on the counterpart subunit of the P-axis dimer (K.Arai et al., unpublished results). In any case, it is likely that the protonated His205 can participate in some interaction network that stabilizes the R state of the enzyme, and thereby stimulates the enzyme activation under acidic conditions. Since substrate pyruvate has a much weaker activation function than Fru(1,6)P2, such a His205-related interaction network may be particularly important in the case of Fru(1,6)P2-independent activation. It is interesting that His205 is well conserved in the enzymes of lactic acid bacteria related to L.casei (Table IV
). We are currently investigating the crucial role of His205 through detailed X-ray crystallography of and additional amino acid replacements in the L.casei enzyme.
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Notes |
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Acknowledgments |
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References |
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Cameron,A.D., Roper,D.I., Moreton,K.M., Mirhead,H., Holbrook,J.J. and Wigley,D.B. (1994) J. Mol. Biol., 238, 615625.[CrossRef][ISI][Medline]
Clarke,A.R., Wigley,D.B., Barstow,D.A., Chia,W.N., Atkinson,T. and Holbrook,J.J. (1987) Biochim. Biophys. Acta, 913, 7280.[ISI][Medline]
Clarke,A.R., Atkinson,T. and Holbrook,J.J. (1989) Trends Biochem. Sci., 14, 101105, 145148.[CrossRef][ISI][Medline]
Dixon,M. and E.C. Webb. (1979) Enzymes, 3rd edn. Longman, London, pp. 400402.
Duncan,M.J. and Hillman,J.D. (1991) Infect. Immun., 59, 39303934.[ISI][Medline]
Eszes,C.M., Sessions,R.B., Clarke,A.R., Moreton,C.K. and Holbrook,J.J. (1996) FEBS Lett., 399, 193197.[CrossRef][ISI][Medline]
Eventoff,W., Rossmann,M.G., Taylor,S.S., Torff,H.-J., Meyer,H., Keil,W. and Kiltz,H.-H. (1977) Proc. Natl Acad. Sci. USA, 74, 26772681.[Abstract]
Ferain,T., Garmyn,D., Bernard,N., Hols,P. and Delcour,J. (1994) J. Bacteriol., 176, 596601.[Abstract]
Fushinobu,S., Kamata,K., Iwata,S., Sakai,H., Ohta,T. and Matsuzawa,H. (1996) J. Biol. Chem., 271, 25611125616.
Fushinobu,S., Ohta,T. and Matsuzawa,H. (1998) J. Biol. Chem., 273, 29712976.
Garmyn,D., Ferain,T., Bernard,N., Hols,P. and Delcour,J. (1995) Appl. Environ. Microbiol., 61, 266272.[Abstract]
Garvie,E.I. (1980) Microbiol. Rev., 44, 106139.[ISI]
Gordon,G.L. and Doelle,H.W. (1976) Eur. J. Biochem., 67, 543555.[Abstract]
Götz,F. and Schleifer,K.H. (1975) Arch. Microbiol., 105, 303312.[ISI][Medline]
Griffin,H.G., Swindell,S.R. and Gasson,M.J. (1992) Gene, 122, 193197.[CrossRef][ISI][Medline]
Hensel,R., Mayr,U., Stetter,K.O. and Kandler,O. (1977) Arch. Microbiol., 112, 8193.[ISI][Medline]
Hensel,R., Mayr,U. and Woenckhause,C. (1983) Eur. J. Biochem., 135, 359365.[Abstract]
Hewitt,C.O., Eszes,C.M., Sessions,R.B., Moreton,K.M., Dafforn,T.R., Takei,J., Dempsey,C.E., Clarke,A.R. and Holbrook,J.J. (1999) Protein Eng., 12, 491496.
Holbrook,J.J., Liljas,A., Steindel,S.J. and Rossmann,M.G. (1975) In Boyer,P.D. (ed.), The Enzymes, 3rd edn. Academic Press, New York, Vol. 11, pp. 191292.
Holland,R. and Pritchard,G.G. (1975) J. Bacteriol., 121, 777784.[ISI][Medline]
Ito,Y. and Sasaki,T. (1994) Biosci. Biotech. Biochem., 58, 15691573.[ISI][Medline]
Iwata,S. and Ohta,T. (1993) J. Mol. Biol., 230, 2127.[CrossRef][ISI][Medline]
Iwata,S., Kamata,K., Minowa,T. and Ohta,T. (1994) Nat. Struct. Biol., 1, 176185.[ISI][Medline]
Kim,S.F., Baek,S.J. and Pack,M.Y. (1991) Appl. Environ. Microbiol., 57, 2413-2417.
Koide,S., Yokoyama,S., Matsuzawa,H., Miyazawa,T. and Ohta,T. (1991) J. Biol. Chem., 264, 86768679.
Koide,S., Yokoyama,S., Matsuzawa,H., Miyazawa,T. and Ohta,T. (1992) Biochemistry, 31, 53625368.[ISI][Medline]
Kunai,K., Machida,M., Matsuzawa,H. and Ohta,T. (1986) Eur. J. Biochem., 160, 433440.[Abstract]
Kunkel,T.A. (1985) Proc. Natl Acad. Sci. USA, 82, 488492.[Abstract]
Laemmli,U.K. (1970) Nature, 227, 680685.[ISI][Medline]
Llanos,R.M., Hillier,A.J. and Davidson,B.E. (1992) J. Bacteriol., 174, 69566964.[Abstract]
Matsuzawa,H., Machida,M., Kunai,K., Ito,Y. and Ohta,T. (1988) FEBS Lett., 233, 375378.[CrossRef][ISI]
Mayr,U., Hensel,R., Deparade,M., Pauly,H.E., Pfleiderer,G. and Trommer,W.E. (1982) Eur. J. Biochem., 126, 549558.[ISI][Medline]
Minowa,T., Iwata,S., Sakai,H., Masaki,H. and Ohta,T. (1989) Gene, 85, 161168.[CrossRef][ISI][Medline]
Monod,J., Wyman,J. and Changeux,J.-P. (1965) J. Mol. Biol., 12, 88118.[ISI][Medline]
Schröder,G., Matsuzawa,H. and Ohta,T. (1988) Biochem. Biophys. Res. Commun., 152, 12361241.[ISI][Medline]
Taguchi,H. and Ohta,T. (1991) J. Biol. Chem., 266, 1258812594.
Taguchi,H. and Ohta,T. (1992) Eur. J. Biochem., 209, 993998.[Abstract]
Taguchi,H. and Ohta,T. (1995) Biosci. Biotech. Biochem., 59, 451458.[ISI][Medline]
Taguchi,H., Matsuzawa,H. and Ohta,T. (1984) Eur. J. Biochem., 145, 283290.[Abstract]
Taylor,S.S. (1977) J. Biol. Chem., 252, 17991806.[ISI][Medline]
Uchikoba,H., Fushinobu,S., Wakagi,T., Konno,M., Taguchi,H. and Matsuzawa,H. (2002) Proteins: Struct. Funct. Genet., 46, 206214.[CrossRef][ISI][Medline]
Wigley,D.B., Gamblin,S.J., Turkenburg,J.P., Dodson,E.J., Piontek,K., Muirhead,H. and Holbrook,J.J. (1992) J. Mol. Biol., 223, 317335.[ISI][Medline]
Wirz,B., Suter,F. and Zuber,H. (1983) Hoppe-Seyler's Z. Physiol. Chem., 364, 893909.[ISI][Medline]
Received July 19, 2001; revised October 3, 2001; accepted October 16, 2001.