Critical Role of Lys212 and Tyr140 in Porcine NADP-dependent Isocitrate Dehydrogenase

Abstract

Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454–43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01–9% of wild type. The most striking change is in the pH-Vmax curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2–0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-Vmax profiles. Vmax is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase. Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454–43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01–9% of wild type. The most striking change is in the pH-Vmax curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2–0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-Vmax profiles. Vmax is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase. Porcine heart mitochondrial NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42), a citric acid cycle enzyme, catalyzes first the oxidation of isocitrate to form enzyme-bound oxalosuccinate, followed by the decarboxylation of the β-keto acid to yield α-ketoglutarate, NADPH, and carbon dioxide. It has been postulated that the reaction is initiated by the deprotonation of the hydroxyl group of isocitrate; the hydrogen of the resultant alkoxide ion would then be easily removed as hydride and transferred to NADP (1.Colman R.F. Pept. Protein Rev. 1983; 1: 41-69Google Scholar). An enzymic general base was proposed as participating in the removal of the proton from the hydroxyl group. Since the pH dependence of Vmax reveals the requirement for the basic form of a group of pKaes 5.7, the general base was initially thought to be a carboxylate side chain of the enzyme (2.Colman R.F. J. Biol. Chem. 1973; 248: 8137-8143Abstract Full Text PDF PubMed Google R.F. PubMed Google Scholar). isocitrate dehydrogenase a for and has been that the pKaes of the of the hydroxyl of isocitrate N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). The decarboxylation of to the form of the and enzymic general acid was proposed to of the at the carbon from which the was the The general acid of the enzyme has not been Porcine NADP-dependent isocitrate dehydrogenase is a of each of which has a of and R.F. PubMed Google R.F. PubMed Google Scholar). The crystal structure of the of this isocitrate dehydrogenase has recently been to a of N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). of the acid side from the a of to the The is to of the and to the hydroxyl of to and to of of the as as to of of the N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). and are close to the carboxyl of isocitrate the of has been by R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). Tyr140 of the is close to the of and Lys212 of the is also in the of The of this and this in the of NADP-specific isocitrate dehydrogenase have not been and that are in isocitrate We the of Lys212 by to and and of Tyr140 by and The results that Lys212 and Tyr140 are for by porcine NADP-dependent isocitrate dehydrogenase. of this has been R.F. J. PubMed Google Scholar). for the for and were by was from from was for of Escherichia coli and were from was from was α-ketoglutarate, and heart were from dehydrogenase and dehydrogenase were from and were from was from were from and were from were from acid of was the from The of a heart NADP-dependent isocitrate dehydrogenase as R.F. PubMed Google R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google R.F. J. PubMed Google R.F. Protein PubMed Google R.F. PubMed Google R.F. PubMed Google Scholar). The to mutant enzymes are as K212Q and K212R and and Y140F and Y140T and Y140E and and Y140K and The are mutants were by at the in the of and of as Tyr140 mutants and and Lys212 mutants and of Wild and wild type and mutant enzymes expressed in coli as were purified by on as R.F. PubMed Google R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google R.F. J. PubMed Google R.F. Protein PubMed Google R.F. PubMed Google R.F. PubMed Google Scholar). dehydrogenase was from the by the and the was to a to isocitrate dehydrogenase from as R.F. PubMed Google Scholar). The enzyme was pH and and at The was determined from R.F. PubMed Google Scholar). The of the enzymes was determined by as R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google R.F. PubMed Google R.F. PubMed Google R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). acid was on of the were a as for isocitrate dehydrogenase R.F. PubMed Google Scholar). 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PubMed Google PubMed Google Scholar). and enzyme was by the of NADP to at The was pH 7.4, and in a of The specific activity was as the of the the Km the of was and the were at the The Km and Vmax values were from of substrate pH of pH dependence of Vmax for each of the enzymes was the pH and pH pH pH of the were in The reaction was and rescue was by of acetic to the reaction Lys212 mutant the reaction was the as at pH In pH pH was Tyr140 mutant the reaction pH was and In as the was pH pH The were at of was by a from that by J. Biol. Chem. Full Text PDF PubMed Google Scholar). was in of the pH of the was to and the of the was to to yield a An of the was added to of in a a The was in a The was then to a of The was from by first the the was then from the as a were and The was in of and the pH of the was to The was and at The of was determined by the as a substrate for as R.F. PubMed Google Scholar). dehydrogenase was pH of to were added to that pH and ammonium R.F. PubMed Google Scholar). The dehydrogenase activity was by the of oxidation of to at and of was and for the of which is to the specific of the from the of J. Biol. Chem. Full Text PDF PubMed Google the of of from by isocitrate was determined a pH 7.4, NADPH, and activity in a of wild type mutant enzyme was added to the and at in a for At the of this of was added to the The was to a of at The was and the was also to the The was from the of An from each was of and the was a was for the of in the of isocitrate dehydrogenase. The of was on the and on the of isocitrate dehydrogenase The of was and expressed in of of and of the wild type and mutant heart mitochondrial NADP-dependent isocitrate were the and the the of the positively charged Lys212 for enzyme was replaced by the neutral and and by the positively charged the of the was replaced by the by hydroxyl for by the of a at position and by the positively charged The porcine enzymes were expressed in coli and as the enzymes are expressed and are as by in the of the of this is close to the of from the acid R.F. PubMed Google Scholar). of each enzyme the of the and that of the enzyme is porcine isocitrate which from the coli isocitrate dehydrogenase in of the first of the to enzymes have in their were The mutant and wild type enzymes have that are at and not that not change in the structure of isocitrate of the of the wild type and mutant enzymes were determined by in mutant enzymes that of wild type that as as is The and of Lys212 specific activity of wild type isocitrate dehydrogenase is N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). In contrast, the specific activities of the Lys212 mutant enzymes are decreased 0.01–9% of wild as in These results indicate that Lys212 has in catalytic The specific activity is by K212R, which the positive of the for wild type and Lys212 mutant NADP-dependent isocitrate and in pH 7.4, in a of and Km values at pH 7.4 were determined by the substrate and the at the and Km values at pH 7.4 were determined by the substrate and the at the and Km values at pH 7.4 were determined by the substrate and the at the and Km values at pH 7.4 were determined by the substrate and the at the The and in pH 7.4, in a of Vmax and Km values at pH 7.4 were determined by the substrate and the at the in a The Km values for and substrate of wild type and Lys212 mutant enzymes are in The Km values for NADP are not in Lys212 mutant the Km values for isocitrate are not altered in the K212Q and It is in the K212R in which positively charged acid is replaced by positively charged that the Km for isocitrate is increased wild type. These results suggest that the of may the of isocitrate in the In is that Lys212 not to the of the enzyme for charged The Km for is increased a in the K212Q and mutants is in the K212R substrate for this enzyme has been to be isocitrate R.F. J. Biol. Chem. Full Text PDF PubMed Google R.F. J. Biol. Chem. Full Text PDF PubMed Google R.F. PubMed Google in the for this substrate be in the Km for isocitrate The pH dependence of Vmax was determined for the wild type and mutant enzymes in to the on the ionizable group by of The of substrate and were to the Km values to the enzymes were the pH and NADP were for wild type, K212Q, and K212Y, whereas 8 and NADP were for the K212R The dependence of the on pH was in is the at a is the and is the constant for the ionizable group of the enzyme-substrate complex. In the of the K212Q and the for was replaced by at to a constant of for K212Q and to for of In this was replaced by The may a of the group is the pH-Vmax for wild type and the Lys212 mutant The Vmax of each mutant enzyme at a pH was expressed as a of in to of the of the for the enzymes that in specific in the pH-Vmax of K212Q and are pH as wild type and K212R mutant Wild type enzyme depends on the deprotonated form of a group of pKaes pKaes is increased to Lys212 is replaced by neutral and to by neutral In contrast, is replaced by positive the pKaes is The values of pKaes and are in These results indicate that a positively charged residue at position 212 lowers the pKaes values of the nearby ionizable group in the enzyme-substrate for the pH-Vmax for wild type and Lys212 mutant of NADP-dependent isocitrate dehydrogenase The activities were and in pH pH pH and pH in activity was the of the and in the activity was the of the and in the The activity was the of the and in the in a The and of Tyr140 that the specific activity of the Tyr140 mutant as at pH 7.4, are reduced to 0.2–0.5% that of wild type Tyr140 also has a in the of isocitrate dehydrogenase. Since be replaced by the group is not the of at this the mutant has the hydroxyl group for the group. specific activity is also for the Y140E and Y140K is at pH 7.4, a carboxylate side chain in the of the isocitrate Tyr140 in the decarboxylation of of NADP and isocitrate for wild type and Tyr140 mutant NADP-dependent isocitrate and in pH 7.4, in a of and Km values were determined at pH 7.4 by substrate and the at the in the and Km values were determined at pH 7.4 by substrate and the at the in the and Km values were determined at pH 7.4 by substrate and the at the in the values were determined at pH The was whereas the were at the in the The and in pH 7.4, in a of Vmax and Km values were determined at pH 7.4 by substrate and the at the in the Km values were determined at pH The was whereas the were at the in the determined in a also the Km values for and of the Tyr140 the Km values for NADP and isocitrate are not that of is that of wild type. the Km for NADP is decreased in the and Y140K that the side chain of and the of the The Km for isocitrate is also decreased in the Y140T and Y140E and the Km for is in Y140E and the Y140K mutant Km for The group of to be for and substrate is a to The most striking change in the Tyr140 mutants is in the pH dependence of as in and B. Vmax values of Y140F and Y140T mutant enzymes are and constant from pH 5.3 to 8 In contrast, for Y140E and the Vmax increases as the pH is decreased These be by a in which Vmax depends on the form of ionizable group in the enzyme-substrate The the be by a of a of the is a of that in wild type enzyme, Tyr140 as a general acid that protonates the substrate after and this in mutant In the of the carboxylate of may the as the of and for the of may as the general of the is the by which exogenous are to the of activity in mutant enzymes PubMed Google Protein 1: PubMed Google PubMed Google R. PubMed Google R. PubMed Google PubMed Google Scholar). in the Y140T mutant be by the addition of acetic to at pH and to a in the activity to at this of the activity of wild type enzyme in pH the specific activity of wild type enzyme to that of the enzyme this is the in the wild type and mutant be that acetic a 106-fold of the activity of the Y140T mutant to wild type The addition of to the Y140T mutant the enzyme at the the addition of to wild type enzyme the specific activity to that of the enzyme to wild type enzyme, to 88-fold of the Y140T mutant that the of is the in the wild type and mutant In contrast, the addition of to the Y140T mutant has on These results that exogenous acetic the enzyme by the side chain of and the enzyme by a general acid at the The Y140F and Y140E enzymes are also to a by addition of acetic the activities of mutants are increased to and respectively, at acetic the and side are to for the exogenous In to acetic acid is the rescue the of pH on the of the of acid in the Y140T mutant that the of acid is as the pH is decreased from pH 7.4 to at pH the activity is to that of wild type at the of acid Since the acetic acid increases from pH 7.4 to that the acetic acid is the rescue The K212Q mutant enzyme is activated by the addition at pH 7.4 of and not to 4-fold at to yield specific activities of and These values and respectively, of the activity of wild type In contrast, the addition of the of has on wild type The of to the K212Q mutant is to the of this to the to the ammonium group of the wild type also not These chemical rescue experiments support the conclusion that the side chain of Tyr140 and the side chain of Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase. We also the pH dependence of chemical rescue of the K212Q mutant enzyme by to in that the is the in chemical that at pH the enzyme at whereas at pH a is The (by at pH enzyme that is of the activity of wild type The in in which the of the base increases that the base and the ammonium form to the chemical rescue of the K212Q mutant of of isocitrate dehydrogenase the in the decarboxylation of isocitrate to form dehydrogenase is also to the of from the of as this substrate is first to the of followed by from the J. Biol. Chem. Full Text PDF PubMed Google Scholar). The a of the of the of the reaction from isocitrate to and as by wild type and mutant for the activities of enzymes in the decarboxylation as as in the The of of by wild type enzyme is that of the decarboxylation of the in the reaction to form and the first in the reaction to form the the of the The mutants for Tyr140 have of the activity of wild type enzyme in the decarboxylation reaction and the the that Tyr140 is required to the form of The mutants for Lys212 also of the activity of wild type enzyme in the Lys212 also a in the of the enzyme to the of the of hydrogen from the J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of Lys212 on the be to positive of by the positively charged mutant enzyme that is the neutral K212Q in the of from the activity of which was R.F. PubMed Google and to a in the Km for in the close to mutant enzyme is in to the reaction in the general of the not the activity of the dehydrogenase activity and activity of wild type and mutant dehydrogenase activity was determined at and activity was at as dehydrogenase activity was determined at and activity was at as to wild to wild for isocitrate dehydrogenase activity of was determined dehydrogenase activity was determined at and activity was at as Vmax for isocitrate dehydrogenase activity of was determined R.F. PubMed Google in a In the porcine mitochondrial NADP-dependent isocitrate dehydrogenase the coli and isocitrate is N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). is a that the a in the structure of the Lys212 and Tyr140 of porcine NADP-specific isocitrate dehydrogenase are of are close to the enzyme-bound as in The group of Lys212 is from the of the of the from the of from the of and from the N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). The group of Tyr140 is from the of the from the of and from the N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). were as for The positively charged was replaced by is in is by is the positive and by a group in as a general Tyr140 was to is in the to the was for the group and to in the to for chemical rescue by exogenous and to and the of charged at position of the purified mutant enzymes the and as wild type enzyme, that their are not altered by The Lys212 mutant enzymes have decreased specific activities for the isocitrate dehydrogenase reaction at pH 7.4, that this residue is for enzyme The K212R mutant the specific the of the positive to the catalytic the Lys212 and isocitrate the not to the of the enzyme for the charged substrate as wild type enzyme, the Km for isocitrate not change in the mutants neutral at this position and is in the positively charged K212R mutant that the Km values for isocitrate and are and this may from from the The most striking in the Lys212 mutant enzymes are in the dependence of Vmax on The activity of wild type enzyme the form of enzyme-substrate group a pKaes of experiments isocitrate R.F. PubMed Google that of isocitrate from pH to the pK of be to the carboxyl of It was recently proposed that the pK of is to the of the hydroxyl proton of isocitrate N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google and this is by the results of of the of the and by the dependence of the pKaes on the ion to the N. B. Grodsky, and R. Colman, in In the the in the pKaes to and in the neutral K212Q and K212Y, respectively, that the positive of lowers the pK of the ionizable group hydroxyl group of to is by the of a pKaes for K212R in which is replaced by positive We R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google results of increased pKaes for the enzyme-substrate and were replaced by the neutral and 7.4, and are and respectively, from the group of enzyme-bound isocitrate N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google to to the of the pK of the hydroxyl group. the Tyr140 mutant enzymes also decreased specific activities at pH 7.4, the of Tyr140 in is and pK is to be for Tyr140 is close to the of isocitrate and have been to the of this substrate by hydrogen that the Km for isocitrate is that of wild type in the Y140F mutant the is and is replaced by and These results suggest that the of the enzyme for isocitrate is decreased by from the results were for the Km for the for is in the mutant The residue to the of the enzyme for close to the position of The porcine isocitrate dehydrogenase was as the a was of the porcine enzyme isocitrate in to the of NADP in the porcine enzyme N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). In the structure of the porcine isocitrate the Tyr140 and the of NADP is 8 this that the of the acid on the Km for NADP is the The most notable in the of the Tyr140 mutant enzymes are the altered pH-Vmax profiles. Tyr140 was replaced by a as and Vmax is of wild and constant from pH 5.3 to the in Vmax pH that have to the deprotonation of the hydroxyl of isocitrate N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). that the reaction is by a the of the to enzymic decarboxylation of oxalosuccinate, to yield the the group of Tyr140 in wild type enzyme as the general acid in this of by a as would the The reaction would be and would have a proposed for Tyr140 is by the activity of the Y140F and Y140T mutants that of wild type in the Tyr140 is required to the form of in the The proton of the group of Tyr140 is from the of isocitrate in the of isocitrate dehydrogenase N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). the of isocitrate is removed by the may to of It has been PubMed Google in the of coli the that the the the to the porcine enzyme In the of the porcine Y140E and Y140K the activity is at neutral pH increases as the pH It may be that the forms of the carboxyl group of acid and the ammonium group of as the general the in the of the decarboxylation reaction by isocitrate dehydrogenase. is also the that chemical rescue by acid is as the pH is decreased from pH 7.4 to and the of acetic acid The of Lys212 in the catalytic reaction be to in the pKaes of a nearby ionizable the of the enzyme-substrate is the of K212Q is that of wild type Lys212 may to stabilize the carbanion at of the that is formed decarboxylation of Lys212 is in to the of may also be to the by Lys212 of the carbanion formed first of the from the form of Lys212 may in the from as of the is notable that the pH dependence of chemical rescue of K212Q by that the base and ammonium of in the and are the in the coli NADP-dependent isocitrate dehydrogenase that to Lys212 and Tyr140 of the porcine of in the coli enzyme have been constructed and in to and PubMed Google J. Biol. PubMed Google Scholar). These that the and in wild type enzyme are to a proton to the carbanion decarboxylation PubMed Google not the pH dependence of Vmax of their mutant The crystal structure of the NADP-specific isocitrate dehydrogenase from B. also a and that in the substrate J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). The structure of the NADP-dependent isocitrate dehydrogenase from has been recently N. Full Text Full Text PDF PubMed Google Scholar). enzyme has a in to the B. and porcine that have each of from of the N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google PubMed Google J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). and from the enzymes the enzyme also has and isocitrate N. Full Text Full Text PDF PubMed Google which are to Tyr140 and Lys212 of the porcine isocitrate dehydrogenase N. R.F. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). rescue of mutant enzymes by added has been to in the of the enzyme PubMed Google Protein 1: PubMed Google PubMed Google R. PubMed Google R. PubMed Google PubMed Google Scholar). It is to a acid that the exogenous the by of the mutant of was as that the of a in the catalytic reaction of that enzyme R. PubMed Google and addition of to the mutant of was to the of as to R. PubMed Google Scholar). In the the Y140T mutant isocitrate dehydrogenase was activated by added by acetic These results are a for Tyr140 as a general the side chain of the mutant be by exogenous acetic The of added acetic acid be in the as the in activity of the Y140E mutant the carboxyl as general chemical rescue of the K212Q mutant by addition of the requirement for the side chain of the of the mutant the of the chemical rescue and the crystal structure of the are the of Tyr140 and Lys212 in the decarboxylation by porcine mitochondrial NADP-dependent isocitrate dehydrogenase. We and for the acid