Roberta F. Colman

Insights from retinitis pigmentosa into the roles of isocitrate dehydrogenases in the Krebs cycle

Here we describe two families with retinitis pigmentosa, a hereditary neurodegeneration of rod and cone photoreceptors in the retina. Affected family members were homozygous for loss-of-function mutations in IDH3B, encoding the β-subunit of NAD-specific isocitrate dehydrogenase (NAD-IDH, or IDH3), which is believed to catalyze the oxidation of isocitrate to α-ketoglutarate in the citric acid cycle. Cells from affected individuals had a substantial reduction of NAD-IDH activity, with about a 300-fold increase in the Km for NAD. NADP-specific isocitrate dehydrogenase (NADP-IDH, or IDH2), an enzyme that catalyzes the same reaction, was normal in affected individuals, and they had no health problems associated with the enzyme deficiency except for retinitis pigmentosa. These findings support the hypothesis that mitochondrial NADP-IDH, rather than NAD-IDH, serves as the main catalyst for this reaction in the citric acid cycle outside the retina, and that the retina has a particular requirement for NAD-IDH.

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (KI) of hGST P1-1 on [BITC] is hyperbolic, with KI = 66 ± 7 μm. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr103 and Cys47 are modified equally. S-Methylglutathione reduces modification of Cys47, indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr103, suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in Km for BITC and binds ANS poorly, whereas Y103F has a normal Km for BITC and Kd for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M(w) of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M(w) of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K(m) value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.

Mechanisms for the oxidative decarboxylation of isocitrate: Implications for control

Abstract Mammalian tissues contain two isocitrate dehydrogenases: the DPN-dependent enzyme which is an oligomeric protein subject to allosteric activation by ADP, and the TPN-specific enzyme which is a monomeric protein not generally considered to be subject to regulation. Yet any controls exerted on the oxidative decarboxylation of isocitrate must necessarily encompass both enzymes since the reactions involve common substrates and metal ions. The two enzymes have been isolated in homogeneous state from porcine cardiac muscle. Similarity in their catalytic mechanisms is indicated by kinetic studies and evidence from chemical modification of enzymic functional groups. No appreciable deuterium isotope effect is observed when isocitrate-2- 2 H is substituted for isocitrate-2- 1 H as substrate for either the DPN- or TPN-dependent isocitrate dehydrogenase. The rates of both reactions are decreased about 5-fold in D 2 O as compared with H 2 O as solvent, implying that a proton transfer (perhaps from the hydroxyl group of isocitrate) may be involved in the rate-determining step. The pH dependence of V max measured at different temperatures suggests that an enzyme carboxylate ion is essential for the activity of both enzymes, although its pK is approximately 5.7 for the TPN enzyme and 6.6 for the DPN enzyme. This conclusion is strengthened by the inactivation of both enzymes by carbodiimide in the presence of nucleophiles. Similarly, chemical modification studies have implicated cysteinyl residues in the function of both enzymes. A single methionyl residue has been shown to be critical for catalytic activity of the TPN enzyme but a comparable residue has not yet been identified for the DPN enzyme. A lysyl residue has been shown to be directly involved only in the DPN enzyme. Mechanisms are proposed to account for catalysis of the oxidative decarboxylation of isocitrate by the two enzymes. Of the four stereoisomers, threo-D s -isocitrate is used as substrate by both isocitrate dehydrogenases. However, the pH dependence of K m for isocitrate suggests that dibasic isocitrate is the actual substrate for the DPN enzyme, whereas tribasic isocitrate is utilized by the TPN enzyme. Both enzymes require a divalent metal ion for activity. Kinetic and binding studies indicate that the TPN enzyme interacts with the preformed metal-tribasic isocitrate complex, whereas the DPN enzyme (depending on the available metal concentration) can either bind sequentially free metal ion followed by free dibasic isocitrate or can bind the preformed metal-dibasic isocitrate complex. Manganous ion produces the highest maximum velocity of each enzyme, but marked differences in the relative effectiveness of other metal ions, such as zinc and magnesium, are apparent. ADP, by binding to the allosteric site of the DPN enzyme, uniquely lowers the K m for dibasic isocitrate and its metal complex. However, other metabolic chelators which affect the distribution of free and metal-bound isocitrate can also indirectly alter the affinity of the substrate for both enzymes. The DPN- and TPN-specific isocitrate dehydrogenases are complementary. Several factors, including changes in the concentrations of different metal ions, subtle variations in pH or fluctuations in the levels of physiological chelators, influence the relative flux through the two pathways of oxidative decarboxylation of isocitrate. These changes may contribute to an alteration in the ratio of DPNH to TPNH in the intracellular environment.

Evaluation by Mutagenesis of the Importance of 3 Arginines in α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2α:1β:1γ. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, α-Arg88, β-Arg99, and γ-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes α, β, and γ subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866–36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with α-R88Q, β-R99Q, or γ-R97Q) were purified to homogeneity yielding enzymes with 2α:1β:1γ subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 μmol of NADH/min/mg were measured, respectively, for WT, β-R99Q, and γ-R97Q enzymes. In contrast, mutant enzymes with normal β and γ subunits and α-R88Q mutant subunit has no detectable activity, demonstrating that, although β-Arg99 and γ-Arg97 contribute to activity, α-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for β-R99Q and γ-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that β-Arg99 and γ-Arg97 are needed for normal ADP activation. In addition, the γ-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal α subunit is required for catalytic activity and α-Arg88 likely participates in the isocitrate site, whereas the β and γ subunits have roles in the nucleotide functions of this allosteric enzyme.

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Abstract Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn 2+ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn 2+ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn 2+ , indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn 2+ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75–80% and 25–45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.

Characterization of the physicochemical and catalytic properties of human heart NADP-dependent isocitrate dehydrogenase

The NADP-dependent isocitrate dehydrogenase of human heart was characterized with respect to molecular size, chemical composition, and catalytic properties. The enzyme appears to exist as a single polypeptide chain with a molecular weight of approximately 53.000, with valine as the N-terminal amino acid. No carbohydrate was detected by staining enzyme on polyacrylamide gels with the periodic acid-Schiff base reagent. The amino acid composition of human heart NADP-specific enzyme is very similar to that of the corresponding enzyme from pig and ox hearts. Statistical analytical methods applied to amino acid compositions of several isocitrate dehydrogenases indicate a strong resemblance between the mammalian heart enzymes but little similarity between the heart enzymes and those isolated from either liver or bacteria. Human heart enzyme cross-reacts with rabbit antibody raised against pig heart NADP-dependent isocitrate dehydrogenase, supporting the suggestion of similarity between these two mammalian enzymes. The apparent Michaelis constant for three- Ds -isocitrate was determined to be 2.2 μM for the human heart enzyme. On the basis of the effect of varying the manganous ion concentration on the apparent Michaelis constant for threo- ds -isocitrate, it is postulated that the metal-isocitrate complex is the actual substrate of the enzyme. Citrate, propanetricarboxylate, and threo- ls -isocitrate are weak competitive inhibitors with respect to isocitrate. The essential functional moieties of the coenzyme binding site were examined by testing coenzyme analogs as competitive inhibitors with respect to NADP. the importance of the 2′-phosphata for coenzyme binding was established. The pH dependence of V for the isocitrate dehydrogenase reaction suggests the requirements of a basic form of an essential ionizable group in the enzyme-substrate complex, exhibiting a pK of 5.5, with a heat of ionization of −1.02 kcal/mol. This pK value increases to 5.9 in the presence of 20% ethanol. These results are consistent with the designation of a carboxyl group as the critical ionizable group. Stability studies on the effect of incubating human heart enzyme in normal serum suggest that the absence of isocitrate dehydrogenase activity in serum following myocardial infarction cannot be attributed to instability alone.

6 Site-Specific Modification of Enzyme Sites

Publisher Summary The application of site-specific modification of enzymes and other proteins has become increasingly common. A wide range of chemical classes is available from which a reagent for exploring a particular enzyme can be selected or designed. Structural similarity to the natural ligand is always desirable to ensure target specificity, but the goal of the investigation must be clear to permit a rational choice of the nature of the reactive group. For studies aimed at identifying amino acid participants in active or regulatory sites, or for those aimed at evaluating the kinetic properties, equilibrium binding, spectral characteristics, or X-ray structure of a stoichiometrically modified enzyme, it is important to choose a reagent type with the potential for forming quantitatively a stable, covalent bond between enzyme and affinity label. The haloketone, fluorosulfonylbenzoyl, and haloacyl derivatives may be most appropriate for these applications because they generally yield products of enzyme nucleophiles, which can be isolated and identified within an amino acid sequence. Affinity labels of this type can be used to provide information regarding the effect of occupying the particular site on the conformation of the enzyme, on the reactivity of other sites, or on subunit interactions.