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Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coliisocitrate dehydrogenase and sequence alignment of porcine withE. coli isocitrate dehydrogenase suggests that the porcine Arg101, Arg110, Arg120, and Arg133 are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K m values for isocitrate, Mn2+, and NADP+ similar to wild type enzyme, indicating that Arg120 is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 μmols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K m values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. TheKm values for Mn2+, but not for NADP+, are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg110 and Arg133normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the Km values for isocitrate and Mn2+; however, the marked decrease ink cat suggests a role for Arg101 in catalysis. The V max of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK aes is similar for the R101Q and R120Q enzymes. In contrast, the pK aes for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg110 and Arg133 normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of theE. coli enzyme. Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coliisocitrate dehydrogenase and sequence alignment of porcine withE. coli isocitrate dehydrogenase suggests that the porcine Arg101, Arg110, Arg120, and Arg133 are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K m values for isocitrate, Mn2+, and NADP+ similar to wild type enzyme, indicating that Arg120 is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 μmols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K m values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. TheKm values for Mn2+, but not for NADP+, are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg110 and Arg133normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the Km values for isocitrate and Mn2+; however, the marked decrease ink cat suggests a role for Arg101 in catalysis. The V max of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK aes is similar for the R101Q and R120Q enzymes. In contrast, the pK aes for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg110 and Arg133 normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of theE. coli enzyme. kilobase pair(s) polymerase chain reaction The mitochondrial NADP-specific pig heart isocitrate dehydrogenase (EC 1.1.1.42) catalyzes the divalent metal ion-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate, and it is considered that the metal-tribasic isocitrate complex is the preferred substrate (1.Colman R.F. Pep. Protein Rev. 1983; 1: 41-69Google Scholar). The enzyme is a homodimer (2.Kelly J.H. Plaut G.W.E. J. Biol. Chem. 1981; 256: 330-334Abstract Full Text PDF PubMed Google Scholar, 3.Bailey J.M. Colman R.F. Biochemistry. 1985; 24: 5367-5377Crossref PubMed Scopus (28) Google Scholar), with a subunit mass of 46,600 Da consisting of 413 amino acids of determined sequence (4.Haselbach R.J. Colman R.F. McAlister-Henn L. Biochemistry. 1992; 31: 6219-6223Crossref PubMed Scopus (45) Google Scholar). A13C-NMR study using specifically enriched isocitrate demonstrated that all three carboxyls of the substrate remain fully ionized from pH 5.5 to 7.5 when bound to the enzyme, although the carboxylates of free isocitrate become protonated over this pH range (5.Ehrlich R.S. Colman R.F. Biochemistry. 1987; 26: 3461-3466Crossref PubMed Scopus (19) Google Scholar). This result could be due to the presence of positively charged groups in the region of the substrate binding site. The first evidence of the importance of arginines in the function of NADP-dependent isocitrate dehydrogenase came from the inactivation of the pig heart enzyme by 2,3-butanedione (6.Ehrlich R.S. Colman R.F. Biochemistry. 1977; 16: 3378-3383Crossref PubMed Scopus (25) Google Scholar). A maximum of four arginines were implicated in catalytic activity and, because isocitrate markedly decreased the inactivation rate, it was suggested that at least some of these residues were at or near the isocitrate binding site. Whereas no crystal structure of a mammalian isocitrate dehydrogenase has yet been determined, the structure of the Escherichia coli NADP-dependent isocitrate dehydrogenase is known (7.Hurley J.H. Thorsness P.E. Ramalingam V. Helmers N.H. Koshland D.E. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8635-8639Crossref PubMed Scopus (208) Google Scholar, 8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google Scholar, 9.Stoddard B.L. Dean A. Koshland Jr., D.E. Biochemistry. 1993; 32: 9310-9316Crossref PubMed Scopus (113) Google Scholar). In this bacterial enzyme, Arg119, Arg129, and Arg153 interact with the carboxylates of isocitrate, and Ser113 is also close to the substrate (8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google Scholar). Alignment of the amino acid sequence of E. coli isocitrate dehydrogenase with that of the porcine enzyme reveals only are amino acids among known to interact with the substrate in the E. coli enzyme. shows the amino acid sequence alignment in this region three enzymes and and the E. coli enzyme. In the porcine enzyme, Arg110 and Arg133 respectively, with the E. coli and Arg101 of the pig isocitrate dehydrogenase is withE. is close to is some in the alignment in the of the region however, Arg120 is the only in this of the porcine enzyme and may be to or of the E. coli enzyme. In this to the roles of the positively charged Arg101, Arg110, Arg120, and Arg133 as of isocitrate and by porcine each of these arginines was mutated to the the for and of the mutant as well as the of the and of these enzymes. A of this has been S. Colman R.F. Protein Sci. 1: Scholar). were by the E. and were from The and as well as K were from polymerase was from were by and and were from and were from or was from and the was from and were by A pig heart mitochondrial NADP-specific isocitrate dehydrogenase was a bacterial the enzyme as a fusion with 1987; PubMed Scopus Google Scholar). This was with a as and was to the wild type enzyme S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). This was in the study for using a A.M. Scholar). The was in the first a of using a and a with site. The was then in the to a in with a with site. The is in the base of and the is at the of the The to mutant enzymes R101Q R110Q R120Q and R133Q The are mutated to The to the region for were and was in a in a reaction of of each and of and were at for for and for respectively, for reaction was with an at for and at for the of reaction were The first was purified by and using the the are from the of the was with of and of K enzyme at for was then using and in was in with m pH at for The was by and the was in and then in pH the the in was with of each and at for The was then and The a of of to was at for and then at for with of was of and enzymes. The was E. coli and on were and the was isolated using the of and the was and the of of was The and of was by sequence using the at the of The for and of wild type enzyme S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google was for mutant enzyme with a E. coli with mutant were at in of was to to and the was for at were by at for and in of m pH 7.4, and were in and by at and for with a in a in The was at for and the was the The fusion was from the of by it of in in a at The been in A. This the E. coli isocitrate is not bound to the S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). with of the was a and the fusion was with maltose as S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). The enzyme was cleaved from the by the fusion with or units/mg fusion at for in the presence of isocitrate and to the cleaved enzyme. The was pH m and and to a with to pig heart isocitrate dehydrogenase from the and The enzyme was by m pH and S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). The that specific activity were and to the as S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google to fusion The S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google has been as this more than enzyme the of from and E. coli and at of were in a in a pH PubMed Scopus Google Scholar). of and and were as S. Colman R.F. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The amino acid of wild type and mutant enzymes were determined using an with an and was using a with a Wild type and mutant enzymes in were to the was with pH and m The were at a of and were proteins, and the for isocitrate were and A The a of and a of as determined and The in the were at A from the for each was was using a of the as a function of and were using a Wild type and mutant in pH and m were for The was from the is the in is the of is the of the in is the of of enzyme 413 for pig heart NADP-dependent isocitrate were at by the of NADP+ from the of at The was NADP+, and and the specific activity is as the of these A subunit of 46,600 (4.Haselbach R.J. Colman R.F. McAlister-Henn L. Biochemistry. 1992; 31: 6219-6223Crossref PubMed Scopus (45) Google was to the of enzyme and the was determined from Colman R.F. 1981; PubMed Scopus Google Scholar). K the of isocitrate or Mn2+, was and the were at the the m of isocitrate and for the K m to and for the NADP+ K m to NADP+ were The K V max values were from the The pH of V max was using the pH pH and pH and at The were all the of isocitrate to the R110Q and R133Q max and K m values for isocitrate were determined at the pH values using isocitrate to the The pH were using isocitrate for the wild type and R120Q isocitrate for R101Q enzyme, and isocitrate for R110Q and R133Q enzymes. these all the enzymes were with A of in was and its pH was to it in the The pH of the was at each the crystal structure of the mammalian enzyme has not yet been determined, were on an using Insight II The sequence alignment was using for of NADP-dependent isocitrate dehydrogenase of pig heart E. coli, and is the as in of R.J. Colman R.F. McAlister-Henn L. Biochemistry. 1992; 31: 6219-6223Crossref PubMed Scopus (45) Google The crystal structure of E. isocitrate dehydrogenase was as the structure (8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google for the Modeler for the structure of the pig heart NADP-dependent isocitrate dehydrogenase. expressed as from the were then The with the of function to the of the generated from a from the alignment was by the The porcine NADP-dependent isocitrate with glutamine for at each of the and were generated using by a J.M. Colman R.F. Biochemistry. 1985; 24: 5367-5377Crossref PubMed Scopus (28) Google Scholar). The first and and the was with the enzymes to the and was it not with the the was the been with K and to with the enzymes. A of from on the been with were and that a were for sequence that the been in all of the mutant and that the of the were The were in of and the was increased from S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google to to the of of the fusion The of in for more fusion than S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). The for of the fusion with was also increased from to these only of fusion This of as compared with the by the S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar). shows that the wild type and mutant enzymes the were as by The subunit of the four mutant is similar to that of wild type enzyme. it that the of the R120Q mutant is to the this is not considered significant because it was only in this and is due to of this The of the porcine and E. coli isocitrate in of the first amino The of these purified wild type and mutant porcine enzymes that are not with the E. coli enzyme. the of the wild type and mutant were to on a with m pH and m The wild type and mutant enzymes all and with an of The results that wild type and mutant isocitrate as in The of the isocitrate was determined using a of for the all exhibit an mass of indicating that the not the of isocitrate dehydrogenase. of the expressed enzymes were to these in the enzyme. The of all are similar to that of the wild type all exhibit at and These results that the not in the structure of these isocitrate dehydrogenases. shows the specific activities of wild type and mutant enzymes as The purified wild type enzyme expressed in E. coli a specific activity, is similar to that of the enzyme isolated from pig heart in S. Colman R.F. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The R101Q and R110Q marked in specific activity, the R133Q mutant is as as wild indicating that the of these three mutant enzymes are The mutant R120Q has a specific activity similar to that of wild for wild type and mutant NADP-dependent isocitrate specific activity and Km values for and the for specific activity was in pH 7.4, with and m values for and NADP+ were determined at pH by the of or NADP+ the at the The were by in a the for specific activity was in pH 7.4, with and m values for and NADP+ were determined at pH by the of or NADP+ the at the The were by four exhibit values of K m for the NADP+, similar to that of wild type enzyme, as in These results that these arginines are not in binding. The R120Q mutant also has m for similar to that of wild type indicating that Arg120 is not in In contrast, the R101Q, R110Q, and R133Q m values for that are increased and respectively, with a role for these arginines in binding. II shows the for the isocitrate, wild and mutant enzymes. The R120Q mutant has V max and K m values similar to of wild type enzyme, indicating that Arg120 is not in the function of the enzyme. The R101Q mutant a cat that is only that of wild and it has a K m for isocitrate that is increased The R110Q and R133Q exhibit cat values that respectively, and that of wild type. K for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. These that the of the enzyme for isocitrate is impaired by Arg110 and The in the catalytic of mutant compared with that of wild type enzyme. Whereas mutant R120Q is similar to that of wild R101Q and R133Q are in compared with that of wild type. The R110Q mutant a decrease in compared with wild because it has a for K m for isocitrate and a lower V of wild type and mutant isocitrate for substrate values are than the specific activities in because the V max values were by to of isocitrate for each the in was the an isocitrate of wild type enzyme, isocitrate is a but that is not the for the R110Q and R133Q because of K m values for values are than the specific activities in because the V max values were by to of isocitrate for each the in was the an isocitrate of wild type enzyme, isocitrate is a but that is not the for the R110Q and R133Q because of K m values for enzyme activity was at pH 7.4, as that the of isocitrate were The were as These values are than the specific activities in because the V max values were by to of isocitrate for each the in was the an isocitrate of wild type enzyme, isocitrate is a but that is not the for the R110Q and R133Q because of K m values for in a The enzyme activity was at pH 7.4, as that the of isocitrate were The were as The pH of V max was determined for the wild type and mutant enzymes from pH to for with the pH for the enzyme isolated from pig R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.F. PubMed Scopus Google Scholar, R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The enzyme has been to be over this pH m values for isocitrate are for some of the mutant that each enzyme is with to isocitrate over the pH range at the of the substrate for that enzyme for the wild type and R120Q for R101Q enzyme, and isocitrate for R110Q and R133Q The of on pH was in with the is the maximum at a is the pH maximum aes is the for the The pK aes values for wild type and mutant enzymes are in of the of the for the the activity of each mutant enzyme at each pH was expressed as a of its maximum shows the of for wild type and mutant enzymes. The in the are the to for using the pK aes values in The max for the mutant R120Q is similar to that of wild type enzyme. mutant R101Q, the pK aes is lower than that of wild type enzyme. In contrast, R110Q and R133Q exhibit increases in pK aes to and respectively, as compared with for wild type enzyme. These results that the positive charges of Arg110 and Arg133normally lower the pK of the nearby catalytic base to facilitate its values for wild type and mutant NADP-dependent isocitrate pK aes values of the enzymes were determined as in a The pK aes values of the enzymes were determined as The bacterial and NADP-dependent isocitrate the catalytic with similar S. McAlister-Henn L. Colman R.F. Protein PubMed Scopus Google Scholar, P.E. Koshland Jr., D.E. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). also the of amino yet the of amino acid is on the sequence alignment of the mammalian and E. coli isocitrate of the amino are in all and of the crystal of the E. coli enzyme, Arg101, Arg110, Arg120, and Arg133 were for by as candidates for with the carboxylates for glutamine was to because it the positive of the wild type amino acid but is and is similar in to It is to a at a in to on the structure of the enzyme. In contrast, McAlister-Henn L. Biochemistry. PubMed Scopus Google Scholar), in of the NADP-dependent isocitrate the negatively charged for and not the of this on the structure of the enzyme. the normally in electrostatic of a of isocitrate, then of by could a electrostatic the enzyme and it to the of the mutant enzymes. In of the to mutant isocitrate activity, that were not McAlister-Henn L. Biochemistry. PubMed Scopus Google Scholar). In contrast, all of to glutamine activity to of a of These that only the R120Q mutant is similar to the wild type enzyme in the impaired function in isocitrate binding or in catalysis. Wild type and mutant pig mitochondrial isocitrate were in the to at on indicating that all the the of the wild type and mutant enzymes are that the not in the structure of the enzymes. The R120Q mutant enzyme has a specific activity, K for isocitrate, Mn2+, and NADP+ and max similar to of wild type enzyme. These that Arg120 is not in or substrate binding. The the of the mammalian and the E. coliisocitrate dehydrogenase is and of the E. coli enzyme the porcine enzyme not an to of the E. coli enzyme that to the of isocitrate (8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google Scholar). the R110Q and R133Q mutant enzymes increases m values for isocitrate and 165-fold, and elevated Km values for and These results that positive charges of Arg110 and Arg133 normally the binding of by electrostatic with the negatively charged of the positive by of the glutamine for the of isocitrate for the enzyme. In the R110Q and R133Q mutant enzymes exhibit of max to yield pK aes values of 6.4 and 7.4, respectively, as compared with 5.5 for the wild type enzyme. R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.F. PubMed Scopus Google Scholar, R.F. J. Biol. Chem. Full Text PDF PubMed Google on the of V max on pH for the pig heart NADP-dependent isocitrate dehydrogenase. This could be by a for of the form of an group of pK the pK increased when the catalytic activity was in a of lower R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), but not when the was from to R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the pK was to of a group in the evidence that isocitrate fully ionized over this pH range (5.Ehrlich R.S. Colman R.F. Biochemistry. 1987; 26: 3461-3466Crossref PubMed Scopus (19) Google Scholar), the pK of be to an The isocitrate dehydrogenase reaction is to be by of a from the of isocitrate to the of a to NADP+ (1.Colman R.F. Pep. Protein Rev. 1983; 1: 41-69Google Scholar, 8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google a base on the enzyme has been to this A study in acids of the pig heart enzyme were by suggests that or as the catalytic base because the and in pK S. Colman R.F. J. Scholar). results on the to glutamine at and that the positive charges of Arg110 and Arg133 normally to lower the pK of when the positively charged arginines are by the nearby a as by the in pK aes of the group in the The in pK aes is in the R133Q than in the R110Q mutant enzyme, to the that Arg133 is than Arg110 to the catalytic base of the enzyme. The R101Q mutant enzyme a decrease ink cat with a m for isocitrate and an m for The max is only as compared with wild type enzyme. The results that Arg101 may a role in catalysis. in porcine Arg101 is with E. is to The activity of E. coli isocitrate dehydrogenase is by with inactivation from of Ser113 P.E. Koshland Jr., D.E. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar, J.H. Dean A.M. Koshland D.E. Stroud R.M. PubMed Scopus Google Scholar, A.M. Koshland D.E. PubMed Scopus Google Scholar, A.M. Koshland Jr., D.E. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). of Ser113 by the negatively charged or also inactivation of the E. coli enzyme due to of the to isocitrate, it has been proposed that electrostatic and the group and the group of isocitrate are the of inactivation in the E. coli enzyme A.M. Koshland D.E. PubMed Scopus Google Scholar). The pig heart enzyme is not by in the is at the to Ser113 in the E. coli enzyme these enzymes be at that site. it is not that of the nearby of isocitrate dehydrogenase to the negatively charged inactivation McAlister-Henn L. Biochemistry. PubMed Scopus Google Scholar), from electrostatic of In study of the porcine enzyme, that Arg101 to the glutamine results in a marked decrease in cat indicating that the positively charged be in the catalytic reaction of isocitrate dehydrogenase. is not yet a structure for mammalian NADP-dependent isocitrate generated an structure for the porcine NADP-specific isocitrate using the Modeler of Insight based on the sequence alignment and the x-ray coordinates of theE. coli enzyme with metal shows the of the the substrate and the four residues for in this Arg120 is from the substrate in this the this group and isocitrate is to the This is with results that the R120Q enzyme is similar to wild with in the specific activity, K m values or pK the isocitrate of the the negatively charged no decrease although is a in the K m for isocitrate, due to electrostatic McAlister-Henn L. Biochemistry. PubMed Scopus Google Scholar). that Arg120 in the porcine mitochondrial isocitrate dehydrogenase is not needed for function. shows Arg110 and Arg133 close to the substrate Arg110 is only to the of isocitrate, in or electrostatic it is to the of to the and that it in electrostatic of This is with the increases in the K m for isocitrate in the R110Q and R133Q with the K m for R110Q in with the more of Arg110 to In the crystal structure of the E. coli enzyme, similar roles been for the Arg153 (8.Hurley J.H. Dean A.M. Koshland D.E. Stroud R.M. Biochemistry. 1991; 30: 8671-8678Crossref PubMed Scopus (242) Google Scholar). The of the pig isocitrate dehydrogenase is also with an of the positively charged Arg110 and Arg133 in the pK of the catalytic to be or S. Colman R.F. J. Scholar). of these arginines are to may strengthen the for this as the catalytic with Arg133 as from the of Arg110 is from the This in is with the in pK a values the positive in the R133Q as compared with the R110Q Arg101 is no than from isocitrate in the and be in binding substrate in the site. This is with the that the R101Q mutant enzyme increases in the K m for isocitrate than the R110Q or R133Q mutants. Arg101 is not close to the proposed enzymic or and with the result that the pK aes is not increased in the R101Q The role of Arg101 be from the It is that at this the positive of to an of the isocitrate that is for the catalytic also be of the for pig isocitrate dehydrogenase that Arg101 is a from the to the at isocitrate is bound in the may this in the may the this in the Arg101 may a role in the reaction from that of a from isocitrate by the catalytic a of a role for Arg101 and the of the The of pig mitochondrial NADP-dependent isocitrate dehydrogenase has to the substrate binding of the enzyme and to the results of The is with most of the Whereas it for an crystal structure of the porcine enzyme, the be for for and for results the structure is determined for of the and for the
Soundar et al. (Tue,) studied this question.