In an effort to reconcile conflicting reports regarding the spectra of the human defense factor hypothiocyanite (OSCN(-)), we have synthesized OSCN(-) by three methods and characterized the product spectroscopically. Method I is lactoperoxidase-catalyzed oxidation of SCN(-) by H(2)O(2) at pH 7. Method II is hydrolysis of (SCN)(2) at pH 13. Method III is oxidation of SCN(-) by OX(-) (X = Cl and Br) at pH 13. All three methods produced essentially the same initial UV, (13)C NMR, and (15)N NMR spectra. The UV spectrum reveals a lambda(max) of 376 nm, which is a previously unreported distinguishing feature. The (13)C NMR spectrum (delta = 127.8 ppm at pH 13 vs dioxane at 66.6 ppm) is comparable to those that have been previously reported for OSCN(-) as prepared by methods I and II (although in some cases different assignments have been made). However, the (15)N NMR spectrum we measure (delta = -80.6 ppm at pH 13 vs NO(3)(-) at 0 ppm) contrasts with previous reports. We conclude that all three methods produce the same species, and the spectra are now self-consistent with the formulation OSCN(-).

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead-end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated as 11 ± 2 mM as measured using Mg 2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg 2+ and α-ketoadipate. Curvature is observed in the double reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases the inhibition changes to uncompetitive consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO 2 , α-ketoadipate and NADH. Isocitrate is a slow substrate with a rate of V/E t 216-fold lower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg 2+ bind to enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg 2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The K eq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.Homoisocitrate dehydrogenase (3-carboxy-2-hydroxyadipate dehydrogenase; EC 1.1.1.87) (HIcDH) 1 catalyzes the fourth reaction of the α-aminoadipate pathway (AAA) for lysine synthesis, the NAD-dependent conversion of homoisocitrate to α-ketoadipate (α-Ka) (Scheme 1) (1). Among the 20 common proteinogenic amino acids, lysine is the only one known to have two diverse pathways for its synthesis (2). In bacteria, plants and lower fungi such as phycomycetes or algal fungi, lysine is synthesized via the diaminopimelate pathway, beginning with the phosphorylation of aspartate by aspartokinase. However, it is synthesized via the α- † This work is supported by a grant from the National Institute of Health GM 071417 (to P. F. C. and A. H. W.), and the Grayce B. Kerr Endowment to the University of Oklahoma (to P. F. C.). *Corresponding author: E-mail: pcook@chemdept.chem.ou.edu Tel: 405−325−4581 Fax: 405−325−7182. 1 Abbreviations: HIcDH, homoisocitrate dehydrogenase; AAA, α-aminoadipate pathway; 6-PGDH, 6-phosphogluconate dehydrogenase; ICDH, isocitrate dehydrogenase; IPMDH, 3-isopropylmalate dehydrogenase; TDH, tartrate dehydrogenase; NAD, nicotinamide adenine dinucleotide (the + charge on the nicotinamide ring is omitted for convenience); NADH, reduced nicotinam...

Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming)) catalyzes the final step in the α-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate α-Kg and lysine using NAD + as an oxidizing agent. Proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD and saccharopine bind, a group with a pK a of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pK a of 7.2 accepts a proton from H 2 O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate α-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V 2 /K NADH , consistent with a steady state random mechanism, and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect of D V 2 and D (V 2 /K Lys ), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effect, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V 2 and V 2 /K Lys suggesting proton transfer exists in at least two sequential transition states.Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming); (EC 1.5.1.7)) (SDH 1 ) catalyzes the final step in the α-aminoadipate pathway (AAA) for the de novo synthesis of L-lysine in fungi (1,2). The enzyme catalyzes the reversible † This work is supported by the Grayce B. Kerr Endowment to the University of Oklahoma (to P.F.C.), and a grant (GM 071417) from the National Institute of Health (to P.F.C. and A.H.W.). * Corresponding author: E-mail: pcook@chemdept.chem.ou.edu 1 Abbreviations: SDH, saccharopine dehydrogenase; AAA, α-aminoadipate pathway; α-Kg, α-ketoglutarate; Sacc,...

(η6-Benzene)(δ/λ-1,1‘-biphenyl-2,2‘-diamine)chlorometal(II) hexafluorophosphate (1; metal = ruthenium, osmium) have been synthesized. The rigid nature of the seven-membered chelate ring formed by the 1,1‘-biphenyl-2,2‘-diamine (dabp) ligand renders the complexes chiral. The resulting C 1 molecular symmetry of 1(M=Ru) that we have observed in the solid state by single-crystal X-ray crystallography is preserved in solution on the NMR time scale. The four N−H protons of 1(M=Ru,Os) are chemically inequivalent in the 1H NMR spectrum at 20 °C. Spin-perturbation NMR experiments in acetone solutions reveal pairwise exchange of the resonances that correspond to the N−H protons on the spin-relaxation time scale. The three mechanisms that would account for such an exchange (atropisomerization of the dabp ligand, inversion of stereochemistry at the metal center, and simultaneous inversion of the stereochemistry at the metal and the ligand) are distinguishable, provided a proper assignment of the four N−H protons can be made in the NMR spectra. Having made that assignment, we conclude from 2D EXSY NMR spectroscopy that the mechanism of exchange is inversion of stereochemistry at the dabp ligand center. This observation contrasts with previous reports that conformational isomers of dabp can be resolved.

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.

Manganese(II) porphyrins are isoelectronic with iron(III) porphyrins, and previously reported work suggests that manganese nitrosyl porphyrins are good structural models for their kinetically unstable and biologically relevant ferric-NO analogues. We have prepared a new set of six-coordinate manganese nitrosyl porphyrins of the general form (por)Mn(NO)(L) (por = TTP, T(p-OCH 3 )PP; L = piperidine, methanol, 1-methylimidazole) in moderate to high yields. The (por)Mn(NO)(pip) complexes were prepared from the reductive nitrosylation of the (por)MnCl compounds with NO in the presence of piperidine. The IR spectra of the (por)Mn(NO)(pip) compounds as KBr pellets show new strong bands at 1746 cm −1 (for TTP) and 1748 cm −1 (for (T(p-OCH 3 )PP) due to the NO ligands. Attempted crystallization of one of these compounds (por = TTP) from dichloromethane-methanol resulted in the generation of the methanol complex (TTP)Mn(NO)(CH 3 OH). Reaction of the (por) Mn(NO)(pip) compounds with excess 1-methylimidazole gave the (por)Mn(NO)(1-MeIm) derivatives in good yields. The IR spectra of these compounds show ν NO bands that are ~12 cm −1 lower than those of the (por)Mn(NO)(pip) precursors, indicative of greater Mn→NO π-backdonation in the 1-MeIm derivatives. X-Ray crystal structures of three of these compounds, namely (TTP)Mn (NO)(CH 3 OH), (TTP)Mn(NO)(1-MeIm) and (T(p-OCH 3 )PP)Mn(NO)(1-MeIm) were obtained, and reveal that the NO ligands in these complexes are linear.

Tryptamine-4,5-dione (1) is formed by oxidation of 5-hydroxytryptamine by reactive oxygen and reactive nitrogen species. Dione 1 is a powerful electrophile that can covalently modify cysteinyl residues of proteins and deactivate key enzymes. Thus, 1 has been suggested to play a role in the degeneration of serotonergic neurons in brain disorders such as Alzheimer's disease or evoked by amphetamine drugs. However, if formed in the brain, it is also likely that 1 would react with low molecular weight thiols such as cysteine (CySH) and glutathione (GSH). The resulting metabolites might not only contribute to the degeneration of serotonergic neurons but also, perhaps, serve as biomarkers of such neurodegeneration. In this investigation, it is shown that in oxygenated buffer at pH 7.4 dione 1 reacts with CySH and other low molecular weight sulfhydryls such as GSH, N-acetylcysteine, and cysteamine to form, first, the corresponding 7-S-thioethers of the dione. However, unlike the glutathionyl and N-acetylcysteinyl conjugates of 1, the 7-S-cysteinyl conjugate is very unstable at pH 7.4 forming a number of novel products, the nature of which are dependent on the relative concentrations of 1 and CySH. These products have been isolated, and spectroscopic and other evidence is provided in support of their proposed chemical structures.

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