Saccharopine dehydrogenase. Substrate inhibition studies.

In the direction of reductive condensation of alpha-ketoglutarate and lysine, saccharopine dehydrogenase (N6-(glutar-2-yl)-L-lysine:NAD oxidoreductase (lysine-forming) is inhibited by high concentrations of alpha-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by alpha-ketoglutarate and lysine is linear uncompetitive versus NADH. However, when the inhibition is examined with alpha-ketoglutarate or lysine as the variable substrate, the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory substrate. These inhibition patterns, the lack of interaction of structural analogs of lysine such as ornithine and norleucine with the E-NAD+ complex (Fujioka M., and Nakatani, Y. (1972) Eur. J. Biochem. 25, 301-307), the identity of values of inhibition constants of alpha-ketoglutarate and lysine obtained with either one as the substrate inhibitor, and the substrate inhibition data in the presence of a reaction product, NAD+, are consistent with the mechanism that substrate inhibition results from the formation of a dead-end E-NAD+-alpha-ketoglutarate complex followed by the addition of lysine to this abortive complex.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.     

Complex I binds several mitochondrial NAD-coupled dehydrogenases

NADH:ubiquinone reductase (complex I) of the mitochondrial inner membrane respiratory chain binds a number of mitochondrial matrix NAD-linked dehydrogenases. These include pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, mitochondrial malate dehydrogenase, and beta-hydroxyacyl-CoA dehydrogenase. No binding was detected between complex I and cytosolic malate dehydrogenase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, lipoamide dehydrogenase, citrate synthase, or fumarase. The dehydrogenases that bound to complex I did not bind to a preparation of complex II and III, nor did they bind to liposomes. The binding of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and mitochondrial malate dehydrogenase to complex I is a saturable process. Based upon the amount of binding observed in these in vitro studies, there is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space. The possible metabolic significance of these interactions is discussed.     

One- and two-electron reduction of ubiquinone homologs by NADH- dehydrogenase preparations from the mitochondrial respiratory chain

The mechanism of ubiquinone homologs reduction by different preparations of mitochondrial NADH dehydrogenase: complex I within submitochondrial particles, isolated NADH-ubiquinone oxidoreductase and soluble low molecular weight NADH dehydrogenase, has been investigated. It has been shown that NADH oxidation via the rotenone-insensitive reaction is associated with one-electron reduction of low molecular weight ubiquinone homologs (Q0, Q1, Q2) to semiquinone with subsequent fast oxidation of the latter by atmospheric oxygen to form a superoxide radical. The two-electron ubiquinone reduction to quinol in the rotenone-sensitive reaction is unaccompanied by the semiquinone release from the enzyme active center into the surrounding solution.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.     

Quantitation of the interaction between citrate synthase and malate dehydrogenase

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Interaction of rhodanese with mitochondrial NADH dehydrogenase

NADH dehydrogenase is an iron-sulfur flavoprotein which is isolated and purified from Complex I (mitochondrial NADH: ubiquinone oxidoreductase) by resolution with NaClO4. The activity of the enzyme (followed as NADH: 2-methylnaphthoquinone oxidoreductase) increases linearly with protein concentration (in the range between 0.2 and 1.0 mg/ml) and decreases with aging upon incubation on ice. In the present work a good correlation was found between enzymic activity and labile sulfide content, at least within the limits of sensitivity of the assays employed. Rhodanese (thiosulfate: cyanide sulfurtransferase (EC 2.8.1.1) purified from bovine liver mitochondria was shown to restore, in the presence of thiosulfate, the activity of the partly inactivated NADH dehydrogenase. Concomitantly, sulfur was transferred from thiosulfate to the flavoprotein and incorporated as acid-labile sulfide. Rhodanese-mediated sulfide transfer was directly demonstrated when the reactivation of NADH dehydrogenase was performed in the presence of radioactive thiosulfate (labeled in the outer sulfur) and the 35S-loaded flavoprotein was re-isolated by gel filtration chromatography. The results indicated that the [35S]sulfide was inserted in NADH dehydrogenase and appeared to constitute the structural basis for the increase in enzymic activity.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]

The activity of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle is controlled by ADP and the reaction products, i. e. succinyl-CoA and NADH. ADP activates the alpha-ketoglutarate dehydrogenase component of the complex, whereas NADH inhibits alpha-ketoglutarate dehydrogenase and lipoyl dehydrogenase. In the presence of NADH the kinetic curve of the complex with respect to alpha-ketoglutarate and NAD and the dependence of upsilon versus [NAD] and upsilon versus [Lip (SH)2] in the lipoyl dehydrogenase reaction are S-shaped. In the absence of inhibitor ADP had no activating effect on lipoyl dehydrogenase; however, in the presence of NADH ADP decreases the cooperativity for NAD. The cooperative kinetics of the constituent enzymes of the complex are indicative of its allosteric properties. Isolation of the alpha-ketoglutarate dehydrogenase complex and its lipoyl dehydrogenase and alpha-ketoglutarate dehydrogenase components in a desensitized state confirms their allosteric nature. It is assumed that NADH effects of isolated alpha-ketoglutarate dehydrogenase is due to a shift in the equilibrium between different oligomeric forms of the enzyme.     

Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for energy consuming processes like the synthesis of ATP. Homologues of this complex exist in bacteria, archaea, in mitochondria of eukaryotes and in chloroplasts of plants. The bacterial and mitochondrial complexes function as NADH dehydrogenase, while the archaeal complex works as F420H2 dehydrogenase. The electron donor of the cyanobacterial and plastidal complex is not yet known. Despite the different electron input sites, 11 polypeptides constitute the structural framework for proton translocation and quinone binding in the complex of all three domains of life. Six of them are also present in a family of membrane-bound multisubunit [NiFe] hydrogenases. It is discussed that they build a module for electron transfer coupled to proton translocation.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Active site water molecules revealed in the 2.1 A resolution structure of a site-directed mutant of isocitrate dehydrogenase

Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refined to 2. 1 A. Several new features were identified relative to the wild-type structure. Seven side-chains previously unplaced in the wild-type structure were identified and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identified.Examination of the K230M active sites (K230M isocitrate and K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates approximately 30 degrees toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identified as a sulfate molecule. Its location and interactions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysine 230; a water molecule in that location in K230M suggests it may influence catalysis in the mutant. Comparison of K230M complexed with isocitrate and alpha-ketoglutarate illuminates the influence a ligand has on active site water structure.     

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.     

Regulation of cooperative properties of alpha-ketoglutarate dehydrogenase by means of thiol-disulfide metabolism

The redox state of two SH-groups per enzyme subunit has been shown to control the cooperative properties of alpha-ketoglutarate dehydrogenase. These thiols oxidized, alpha-ketoglutarate dehydrogenase does not exhibit any cooperative properties. The enzyme reduction leads to subunit interactions. It has been found that the most effective agent reducing the alpha-ketoglutarate dehydrogenase thiols essential for the cooperativity is dihydrolipoate, one of the intermediates of the overall alpha-ketoglutarate dehydrogenase reaction. The possibility of changing the properties of alpha-ketoglutarate dehydrogenase in the multienzyme complex under the conditions when the lipoic acid integrated into the complex is reduced, has been investigated. Thus, incubation of the alpha-ketoglutarate dehydrogenase complex with NADH has been found to induce the conversion from the non-cooperative form to the cooperative one, presumably through the reduction of lipoic acid bound to the complex in the reaction catalyzed by lipoyl dehydrogenase, the third component of the complex.     

One-step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep-tag affinity chromatography.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the C-terminus of NuoF or NuoG. Fusion of this peptide to the N-terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.     

Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.