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Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA
Reprint requests to: Roberta F. Colman, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA; e-mail: rfcolman{at}udel.edu; fax: (302) 831-6335.
(RECEIVED August 31, 2004; FINAL REVISION September 27, 2004; ACCEPTED September 28, 2004)
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Abstract |
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-carboxylate of enzyme-bound isocitrate. We used site-directed mutagenesis to replace each of these residues by Ala and Asp. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. All the enzymes retain their native dimeric structures and secondary structures as monitored by native gel electrophoresis and circular dichroism, respectively. Vmax of the three alanine mutants is decreased to 24%–38% that of wild-type enzyme, with further decreases in the aspartate mutants. For T78A and S95A mutants, the major changes are the 10- to 100-fold increase in the Km values for isocitrate and Mn2+. The results suggest that Thr78 and Ser95 function to strengthen the enzyme’s affinity for Mn2+-isocitrate by hydrogen bonding to the -carboxylate of isocitrate. For the Asn97 mutants, the Km values are much less affected. The major change in the N97A mutant is the increase in pKa of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate from 5.23 in wild type to 6.23 in the mutant enzyme. The hydrogen bond between Asn97 and the -carboxylate of isocitrate may position the substrate to promote a favorable lowering of the pK of the enzyme–isocitrate complex. Thus, Thr78, Ser95, and Asn97 perform important but distinguishable roles in catalysis by porcine NADP-specific isocitrate dehydrogenase. Keywords: isocitrate dehydrogenase; citric acid cycle; dehydrogenase
Abbreviations: PCR, polymerase chain reaction • IPTG, isopropylthio-
-D-galactopyranoside • TEMED, N,N,N',N'-tetramethylethylenediamine • SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate • IDH, isocitrate dehydrogenase
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041091805.
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-ke-toglutarate, carbon dioxide, and NADPH. By producing NADPH, the mammalian enzyme has been shown to play an important role in protecting cells against damage from reactive oxygen species (Jo et al. 2001). Inactivation of this isocitrate dehydrogenase by peroxynitrite (Lee et al. 2003) or by lipid peroxidation products leads to substantial decrease in the cellular defense against oxidative stress (Yang et al. 2004). Recently it has been demonstrated, in a rat model of spontaneous hypertension, that decreased activity of mitochondrial NADP-specific isocitrate dehydrogenase closely precedes the development of cardiac hypertrophy, and is associated with modification of the enzyme by 4-hydroxynonenal, the major lipid peroxidation product formed during oxidative stress (Benderdour et al. 2003); however, the site(s) of modification has not been determined. It is important to identify the normal amino acid participants in the catalytic function of isocitrate dehydrogenase in order to understand the significance of post-translational modification of the enzyme by oxidative stress-related compounds.
The NADP-isocitrate dehydrogenase is a dimer of identical subunits, each of which has a molecular mass of 46,600 Da (Bailey and Colman 1985; Haselbeck et al. 1992). The first crystal structure of a mammalian IDH, the porcine mitochondrial NADP-specific isocitrate dehydrogenase complexed with Mn2+ and isocitrate, has recently been reported (Ceccarelli et al. 2002), stimulating the evaluation by site-directed mutagenesis of the functions of amino acids close to the substrate site. Thus, Asp252 and Asp275 were identified as ligands to the required divalent metal ion (Grodsky et al. 2000; Huang et al. 2004). Tyr140 has been implicated as the general acid that protonates the substrate following decarboxylation, and the positively charged Lys212 as an amino acid required to lower the pK of the nearby ionizable group (Kim et al. 2003). In addition, Arg101, Arg110, and Arg133, which are close to the - and -carboxylate groups (Ceccarelli et al. 2002), have been shown to be important contributors to the binding of isocitrate, probably by electrostatic interaction with the negatively charged substrate (Soundar et al. 2000).
Examination of the crystal structure of the porcine NADP-isocitrate dehydrogenase (Ceccarelli et al. 2002) reveals that Ser95, Asn97, and Thr78 are within hydrogen-bonding distance of the oxygens of the -carboxylate of enzyme-bound isocitrate, as illustrated in Figure 1
. These three amino acids are conserved in the amino acid sequences of NADP-dependent isocitrate dehydrogenases from human, pig, rat, mouse, yeast, Escherichia coli, and Bacillus subtilis. 13C-NMR studies of substrate complexes of porcine NADP-isocitrate dehydrogenase reveal constant 13C resonances for all three carboxyls of enzyme-bound isocitrate over the pH range 5.5–7.5, although for free isocitrate there is a considerable change in the chemical shifts for the three carboxyls over the same pH range. These results indicate that the pKs of isocitrate’s carboxyls are lowered when the substrate is bound to the enzyme (Ehrlich and Colman 1987). Hydrogen bonding with one or more enzymic amino acids may stabilize the ionized form of the -carboxyl group of isocitrate and strengthen the affinity of isocitrate for the enzyme. We now assess the importance of Ser95, Asn97, and Thr78 to enzyme function by replacing each of these amino acids by the small, neutral alanine, which cannot participate in hydrogen bonding. In addition, we substituted the negatively charged aspartate for each of these amino acids to test for proximity, in solution, between the negatively charged isocitrate and amino acid side chains at positions 95, 97, and 78.
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demonstrates that the wild-type and six different mutant proteins were well expressed, and each exhibits a single subunit band upon SDS-PAGE; the apparent subunit molecular mass is ~45,000 Da and is the same for wild-type and mutant proteins. N-Terminal sequencing of the individual preparations revealed a single amino acid at each cycle. Since the E. coli and porcine isocitrate dehydrogenases differ in nine of the first 10 amino acids, it was readily determined that all of the E. coli enzyme had been removed, and the final preparations contained homogeneous porcine NADP-specific isocitrate dehydrogenase.
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-carboxylate of enzyme-bound isocitrate and are close enough (at amino acid positions 95, 97, and 78) to do so (see Fig. 1
). Whether hydrogen bonding of substrate to these amino acid side chains is important to enzyme function is tested by substitution of the small, non-hydrogen-bonding amino acid alanine in the mutant enzymes S95A, N97A, and T78A. Table 1 records the kinetic parameters measured at pH 7.4. The Vmax values of these three alanine mutants are decreased to 24%–38% that of the wild-type enzyme, indicating only a small effect on the catalytic rate produced by eliminating hydrogen bonding at these positions. The E. coli isocitrate dehydrogenase is regulated by covalent phosphorylation of Ser113, and the inactivation produced by phosphorylation can be mimicked by substituting aspartate for Ser113, although this does not provide for the possibility that the phosphoserine could be doubly charged (Dean and Koshland 1990; Hurley et al. 1990). Ser113 of the E. coli isocitrate dehydrogenase can be aligned with Ser95 of the porcine enzyme. Although phosphate has not been detected in the mammalian isocitrate dehydrogenase, aspartate was substituted for Ser95 in the porcine enzyme to test the effect of introducing a negative charge at this position. The Vmax of the S95D mutant is dramatically decreased to 0.2% of that of the wild-type enzyme. This large decrease is likely due to electrostatic repulsion between the negatively charged -carboxylate of isocitrate and of the enzyme’s aspartate at position 95. Introduction of an aspartate at position 97 causes Vmax to fall to 1.2% of the wild-type value, undoubtedly resulting also from electrostatic repulsion. However, substitution of aspartate at position 78 results in a Vmax that is only decreased to 17% of that of wild-type enzyme. A negative charge near the substrate binding site is clearly detrimental to the rate of enzyme-catalyzed oxidative decarboxylation, but the magnitude of the effect varies with the location of the negative charge.
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also compares the Km values for NADP, isocitrate, and Mn2+ of wild-type and mutant enzymes. The mutations at positions 95, 97, and 78 cause little perturbation of the Km for coenzyme, indicating that these three amino acids are not critical for binding NADP. In contrast, substitution of alanine for Ser95 or Thr78 results in about a 10-fold increase in the Km for isocitrate, and the effects on this Km are even greater in S95D and T78D. There are corresponding increases in the Km for Mn2+, as might be expected since Mn2+-isocitrate is considered to be the preferred substrate for the NADP-dependent isocitrate dehydrogenase (Colman 1972, 1983; Villafranca and Colman 1972; Ehrlich and Colman 1976).
Replacement of asparagine by alanine at position 97 produces a smaller increase in the Km for isocitrate and for Mn2+, suggesting that hydrogen bonding of Asn97 and the -carboxylate of isocitrate makes only a minor contribution to the binding of isocitrate. Electrostatic repulsion of substrate in the N97D mutant results in a much larger increase in the Km values for isocitrate and for Mn2+.
pH dependence of Vmax
It has been shown that, when the enzyme is fully saturated with NADP, isocitrate, and Mn2+ over the entire pH range tested, Vmax depends on an ionizable group of pK 5.24 (Huang et al. 2004). This pK has been attributed to the deprotonation of the Mn2+-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase (Huang et al. 2004). Figure 3 shows the pH dependence of Vmax for wild-type and the three alanine mutants. The data shown were obtained using 8 mM isocitrate, 4 mM Mn2+, and 2 mM NADP to ensure the enzymes were saturated with coenzyme and substrate over the entire range tested. The same velocities and pK values were obtained when the concentrations were raised to 16 mM isocitrate, 8 mM Mn2+, and 4 mM NADP, demonstrating that the data shown in Figure 3 actually represent Vmax values. The dependence of Vmax, obs on pH was analyzed in accordance with the equation
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for wild-type and all alanine-substituted mutant enzymes, where Vmax, obs is the experimentally observed maximum velocity at a given pH, Vmax, int is the intrinsic pH-independent maximum velocity, and Ka is the dissociation constant for the ionizable group of the enzyme–substrate complex. The Vmax of each mutant enzyme at a particular pH was expressed as a fraction of its own intrinsic maximum velocity, (Vmax, obs)/(Vmax, int), in order to allow comparisons of the shapes of the curves for the several enzymes that differ in specific activity. As shown in Figure 3, the curves for wild-type and the S95A mutant enzyme are almost identical, and that for the T78A enzyme is shifted only slightly toward higher pH. However, the N97A mutant enzyme exhibits a marked increase in the pKa for the enzyme–substrate complex. The pKa and Vmax, int values are summarized in Table 2. The N97A mutant enzyme has a pKa (6.23) that is increased by one logarithmic unit beyond that of wild-type enzyme (5.23). Elimination of the hydrogen bonding between Asn97 and the -carboxylate of isocitrate must have the indirect effect of raising the pK of the nearby Mn2+-coordinated hydroxyl group of isocitrate.
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shows that the circular dichroism spectra of wild-type isocitrate dehydrogenase, as well as the alanine and aspartate mutants at positions 78, 95, and 97, are essentially superimposable, with double minima being observed at 222 and 209 nm. The results indicate that these mutant enzymes do not have appreciable change in their secondary structures.
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(last column) indicate that all the mutant enzymes exhibit molecular masses comparable to those of wild-type enzyme; since all appear to be dimers, changes in the state of oligomerization cannot account for the marked alterations in the kinetic parameters of these mutant enzymes. |
Discussion |
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, the hydroxyl groups of Thr78 and Ser95 are, respectively, 2.57 Å and 2.56 Å from the closest oxygen of the -carboxylate of isocitrate; while the amido nitrogen of Asn97 is 2.83 Å from the closest oxygen of isocitrate’s -carboxylate. All of these amino acid side chains are within hydrogen-binding distance of the isocitrate. To evaluate the role of hydrogen bonding, Thr78, Ser95, and Asn97 were each replaced by alanine. In the case of threonine and serine, alanine is similar in size to the amino acid it replaces; but in substituting alanine for asparagine, a much smaller side chain is placed at position 97 and the -CH3 side chain of alanine will not be close to the bound isocitrate. Furthermore, all three amino acids were replaced by aspartic acid. At position 97, the negatively charged replacement is similar in size to the original asparagine, so this substitution clearly tests the effect of placing a negative charge at 97. In contrast, because aspartate is larger than serine or threonine, substitution by aspartate at positions 95 and 78 causes crowding in the region in addition to introducing negative charge.
Sequence alignment of the dimeric isocitrate dehydrogenase from the pig with those from E. coli and B. subtilis indicates that there is only ~16%–17% identity. Despite the low level of homology, Ser95, Thr78, and Asn97 are conserved; this point is best seen in the structure-corrected sequence alignment of Ceccarelli et al. (Fig. 3 in Ceccarelli et al. 2002). It seems likely that the relatively few conserved amino acids have a significant function for isocitrate dehydrogenase. Even in the monomeric isocitrate dehydrogenase from Azotobacter vinelandii (Yasutake et al. 2002), the serine and asparagine are conserved, although they appear at positions 132 and 135, respectively; the crystal structure of this monomeric isocitrate –Mn2+–IDH complex shows that hydrogen bonds are formed between the substrate and Ser132 and Asn135.
The E. coli isocitrate dehydrogenase is covalently regulated by enzyme-catalyzed phosphorylation of its Ser113 (Dean and Koshland 1990; Hurley et al. 1990). Almost complete inactivation accompanies the phosphorylation, but there is minimal change in the conformation of the protein. The authors concluded that the inactivation upon phosphorylation is caused by electrostatic repulsion, steric hindrance, and (to a lesser extent) loss of hydrogen bonding to the isocitrate (Dean and Koshland 1990). The B. subtilis isocitrate dehydrogenase also has an Asn, Ser, and Thr that are hydrogen-bonded to the corresponding oxygens of the citrate with which it was crystallized (Singh et al. 2001). The equivalent Thr96 of the B. subtilis enzyme is closer to citrate than the distance between isocitrate and the corresponding threonine in the E. coli enzyme, and the distance measured in the B. subtilis enzyme is actually the same as that found for porcine isocitrate dehydrogenase between Thr78 and the closest oxygen of the -carboxylate of isocitrate (Fig. 1). The B. subtilis enzyme has not been found to be phosphorylated in vivo (Singh et al. 2001), although it has been shown in vitro to act as a very poor substrate of the isocitrate dehydrogenase kinase/phosphatase, yielding partially inactivated IDH in which Ser104 is phosphorylated (Singh et al. 2002). There is no evidence that this type of covalent regulation normally occurs in B. subtilis. Similarly, in the mammalian NADP-dependent isocitrate dehydrogenase that was originally isolated from porcine hearts, no phosphorylated enzyme has been detected either by chemical analysis or 31P NMR spectroscopy (Mas and Colman 1984).
In the present study on the recombinant porcine NADP-specific isocitrate dehydrogenase, the major change upon replacing either Ser95 or Thr78 by alanine is to increase 10-to 100-fold the Km for isocitrate and Mn2+. This adverse effect on the affinity of the enzyme for its substrate must reflect the lost contribution toward isocitrate binding of the hydrogen bonds between Thr78 and Ser95 and the isocitrate substrate. There are also small decreases in Vmax for S95A and T78A as compared to wild-type enzyme; these probably indicate that, in the absence of the correct hydrogen bonds to the -carboxylate, isocitrate is less than optimally oriented on the enzyme to participate in oxidative decarboxylation. Replacement of these neutral amino acids by the negatively charged aspartate causes more drastic effects, which must be due to electrostatic repulsion and steric hindrance, as well as to loss of hydrogen bonding. In the case of S95D, Vmax is only 0.2% that of wild-type enzyme, and the Km for isocitrate is increased 277-fold. This change illustrates the effect on activity that could be produced by phosphorylating Ser95, although there is no evidence that such a reaction occurs in vivo.
Substitution of Asn97 by alanine has little influence on the Km for substrates. Since this substitution simply measures the effect of elimination of a hydrogen bond between as-paragine’s amide moiety and the -carboxylate of isocitrate, it is evident that this longer hydrogen bond (2.83 Å) makes only a minor contribution to the enzyme’s affinity for its substrate. The predominant kinetic defect in the N97A mutant is a substantial increase (by one logarithmic unit) in the pK for the ionizable group in the enzyme–substrate complex. This pK has been attributed to the ionization of the metal-liganded hydroxyl of isocitrate bound to the enzyme (Huang et al. 2004). The normal pK of 5.23 of wild-type enzyme is considerably lower than would be expected for a metal-hydroxyl that is not bound to enzyme; this low pK depends on the proximity of the metal-liganded hydroxyl of bound isocitrate to the positively charged Arg101, Arg110, Arg133, and Lys212 in the region of the active site of isocitrate dehydrogenase (Huang et al. 2004). Although Asn97 is relatively far (6.15 Å) from the ionizable –OH, of isocitrate, the hydrogen bond between Asn97 and the -carboxylate of isocitrate must maintain the proper orientation of isocitrate within the active site, so that the ionization of isocitrate’s –OH is subject to the pK-lowering influence of the positively charged amino acids. When that hydrogen bond is eliminated, as in the N97A mutant, the pK of the metal-liganded isocitrate hydroxyl increases to 6.23. It is notable, however, that once the enzyme-bound isocitrate is fully ionized, as shown in the specific activity measured at pH 7.4 or reflected in the intrinsic pH independent maximum velocity, the rate of the isocitrate dehydrogenase reaction is decreased only to ~20% of that of the wild-type enzyme.
The catalytic characteristics of these mutant enzymes indicate that the normal function of Ser95 and Thr78 in porcine NADP-dependent isocitrate dehydrogenase is to enhance the binding strength of the Mn-isocitrate substrate by forming hydrogen bonds with the -carboxylate of isocitrate. The normal role of Asn97, by hydrogen bonding with the -carboxylate of isocitrate, is to properly position the isocitrate within its binding site so that the proton of the C2-hydroxyl can readily be removed prior to the transfer of hydride to NADP (Colman 1983). Thus, Ser95, Thr78, and Asn97 perform important functions in the normal oxidative decarboxylation catalyzed by the mammalian NADP-dependent isocitrate dehydrogenase.
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-chymotrypsinogen A, rabbit muscle enolase, rabbit muscle phosphoglycerate mutase, and chicken egg white lysozyme; while rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was obtained from Boehringer Manheim. Triethanolamine-HCl, NADP, and DL-isocitrate were obtained from Sigma Chemical Co.; and manganese sulfate, sodium sulfate, maltose, acrylamide/bisacrylamide mixture, and sodium acetate were from Fisher Scientific Co. Amicon YM-10 filtration membranes were from Millipore. Human plasma thrombin was obtained from Enzyme Research Laboratory, Inc.; and TEMED, glycine, and Protein Assay Dye Reagent were from BioRad Laboratories. Oligonucleotides for PCR and sequencing primers were synthesized by Bio-synthesis, Inc. The QuikChange XL Site-directed Mutagenesis Kit, purchased from Stratagene, was used to generate the mutant plasmid DNA. For isolation of the plasmid DNA, QIAprep Spin Miniprep kit from QIAGEN was used. New England Biolabs supplied the E. coli TB1 cells and the amylose resin. Other chemicals were purchased from Sigma Chemical Co. or Fisher Scientific.
Site-directed mutagenesis
The 7-kb plasmid harboring a 1.2-kb cDNA-encoding pig heart NADP-dependent isocitrate dehydrogenase (IDP1) was cloned into vector pMAL-c2 (pMALcIDP1), as previously described (Soundar et al. 1996, 2000; Grodsky et al. 2000; Kim et al. 2003; Huang et al. 2004). The oligonucleotides for generating mutant enzymes using the QuikChange XL Site-Directed Mutagenesis Kit were: S95A (forward primer, 5'-GAAGATGTGGAAGGCTCCCAATGGAACCATCCGGAAC-3'; reverse primer, 5'-GGTTC CATTGGGAGCCTTCCACATCTTCTTCAGCTTGAAC-3'); N97A (forward primer, 5'-GGAAGAGTCCCGCTGGAACCATCCG GAACATCC-3'; reverse primer, 5'-CCGGATGGTTCCAGCGG GACTCTTCCACATCTTC-3'); T78A (forward primer, 5'-GTGC CACCATCGCCCCCGATGAGGCCCGTG-3'; reverse primer, 5'-GCCTCATCGGGGGCGATGGTGGCACACTTG-3'); S95D (forward primer, 5'-GAAGATGTGGAAGGATCCCAATGGAA CCATCCGGAAC-3'; reverse primer, 5'-GGTTCCATTGGG ATCCTTCCACATCTTCTTCAGCTTGAAC-3'); N97D (forward primer, 5'-GAAGAGTCCCGATGGAACCATCCGGAA CATC-3'; reverse primer, 5'-GGATGGTTCCATCGGGACTCT TCCACATCTTC-3'); T78D (forward primer, 5'-GTGCCAC-CATCGACCCCGATGAGGCCCGTG-3'; reverse primer, 5'-GCCTCATCGGGGTCGATGGTGGCACACTTG-3'). The underlined codons are mutated sequences. All mutants were confirmed by nucleotide sequence analysis at the DNA Sequencing Facility in the College of Agriculture and Natural Resources, University of Delaware, using as primers: (forward primer, 5'- GCCGACCAGAGGATCAAGG-3'; reverse primer, GTCTTTG AAGCGCCCGTCGTAGGC-3').
Expression and purification of wild-type and mutant enzymes
The recombinant wild-type and mutant enzymes, expressed in E. coli as maltose-binding fusion proteins, were purified by chromatography on an amylose column as previously described (Soundar et al. 1996). The fusion protein of maltose-binding protein and isocitrate dehydrogenase was expressed by adding 0.4 mM IPTG to the 8-L culture broth of E. coli TB1 cells that had been grown at 37°C to A600 nm = 0.4–0.6. Subsequently, incubation was continued for 24 h at 25°C. The cells were collected and suspended in 200 mL of cold 0.02 M triethanolamine chloride buffer (pH 7.4) containing 10% glycerol, 0.2 M Na2SO4, and 2 mM MnSO4 (Buffer A). The suspended cells were frozen at –80°C. After thawing, the cells were lysed by sonication at 20 kHz, 475 W for 10 min for each 100-mL cell suspension. The lysates were centrifuged at 15,000g for 10 min, and the supernatant was applied to the amylose resin, which had been equilibrated in Buffer A. This column separates E. coli isocitrate dehydrogenase from the fusion protein. The fusion protein was eluted with 10 mM maltose-containing Buffer A. Isocitrate dehydrogenase was cleaved from the maltose-binding protein by incubating the fusion protein with human plasma thrombin (41 Units/mg fusion protein) at 25°C for 48 h for wild-type and 24 h for mutant enzymes in the presence of 4 mM isocitrate and 2 mM MnSO4 to stabilize the cleaved enzymes. The digest was dialyzed against 20 mM triethanolamine chloride buffer (pH 7.8) containing 10% glycerol (Buffer B) and applied to a DE52 column, which had been equilibrated in Buffer B to separate isocitrate dehydrogenase from the maltose-binding protein and thrombin, as described previously (Lee and Colman 2002). The fractions of high specific activity were pooled and concentrated to >1 mg/mL, followed by dialysis against 0.1 M triethanolamine chloride buffer (pH 7.7) containing 0.3 M Na2SO4 and 10% glycerol. The enzymes were stored at –80°C.
The purity of the enzymes was evaluated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, as described previously (Soundar et al. 1996). N-terminal amino acid sequencing was conducted using the Applied Biosystem Procise protein/peptide sequencer. The protein concentration was determined from E1%280 nm = 10.8 (Johanson and Colman 1981).
Circular dichroism of the enzymes
Circular dichroism spectra were determined using a Jasco model J-710 spectrophotometer, as described (Grodsky et al. 2000). The enzymes were measured at ~0.25 mg/mL in 25 mM triethanol-amine chloride buffer (pH 7.7) containing 10% glycerol and 75 mM Na2SO4. The final concentration of the enzymes was measured by a Coomassie Blue dye-binding assay, which is based on the Bradford method (Bradford 1976), using wild-type isocitrate dehydrogenase as the protein standard.
Molecular mass determination of the enzymes
The molecular mass of the enzymes was determined by native gel electrophoresis, in accordance with the method of Hedrick and Smith (1968), using polyacrylamide gels from 5% to 10% at a pH of 5.5, as described previously (Lee and Colman 2002; Kim et al. 2003). The standard proteins used were glyceraldehyde-3-phosphate dehydrogenase, 72 kDa; enolase, 84 kDa; phosphoglycerate mutase, 66 kDa; lysozyme,14.3 kDa, and -chymotrypsinogen A, 25.7 kDa.
Enzyme assays and kinetic parameter determinations for the enzymes
Enzyme assays were performed by following at 340 nm the time-dependent reduction of NADP to NADPH. The standard assay solution was 30 mM triethanolamine chloride buffer (pH 7.4), 0.1 mM NADP, 4 mM DL-isocitrate, and 2 mM MnSO4 in a total volume of 1 mL. The specific activity was calculated as the micromoles of NADPH produced per minute per milligram of protein at 25°C under the standard assay condition.
For the Km determinations, the concentration of either isocitrate, NADP or Mn2+, was varied, and the other substrates were maintained at the standard assay conditions unless indicated otherwise.The Km and Vmax values were calculated, along with the standard errors, from direct plots of velocity versus substrate concentration using SigmaPlot software.
pH-rate profile for the enzymes
The pH dependence of Vmax was measured using the following buffers: pH 5.0–6.2, sodium acetate; pH 5.8–7.4, imidazole chloride; pH 6.8–8.0, triethanolamine chloride. All the buffers were 30 mM in anion concentration. The reaction rate was measured using 2 mM NADP, 8 mM isocitrate, and 4 mM MnSO4, unless indicated otherwise.
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Acknowledgments |
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References |
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———. 1983. Aspects of the structure and function of the isocitrate dehydrogenases. Pept. Protein Rev. 1: 41–69.
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  Y. C. Huang and R. F. Colman Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase J. Biol. Chem., August 26, 2005; 280(34): 30349 - 30353. [Abstract] [Full Text] [PDF]
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), T78A (
), and N97A (
). The activities were measured in various buffers (sodium acetate buffer at pH 4.9–6; imidazole chloride buffer at pH 6–7; triethanolamine chloride buffer at pH 7–7.8). For the pH-rate profile, the activity was measured using 4 mM Mn2+, 8 mM isocitrate, and 2 mM NADP in order to ensure saturation of the enzyme over the entire pH range.
), S95A (
), N97A (
), T78D (
), and N97D (
). The CD was measured at pH 7.7 and room temperature, and the molar ellipticity was calculated as described previously (