Major Rate-determining Step Research Articles

Elucidating reaction mechanisms in the oxidative depolymerization of sodium lignosulfonate for enhancing vanillin production: A Density Functional Theory study

Lignin is a vital organic constituent of plant biomass and it can be transformed into value-added chemicals through chemical processes. This study employs the Density Functional Theory (DFT) to investigate the mechanistic effects of various oxidants on the depolymerization of sodium lignosulfonate model compounds into vanillin. Under alkaline conditions, OH- ions participation in the hydrolysis of β-O-4 bonds has been predicted. The dehydration of dihydroconiferyl alcohol to form eugenol, with an energy barrier of 69.0 kcal/mol was identified as the major rate-determining step in the oxidation reaction. In the absence of an oxidant, the energy barrier for the oxidation of eugenol to vanillin was 77.8 kcal/mol. However, when using nitrobenzene or H2O2 as oxidants, the energy barrier has significantly decreased to 12.6 kcal/mol and 21.4 kcal/mol, respectively. The effects of temperature and pressure on the rate-determining step of the reaction were analyzed using Statistical Product and Service Solutions (SPSS) software. These results have demonstrated that the energy barrier for the hydrolysis reaction's rate-determining step is positively correlated with temperature but negatively correlated with pressure. These theoretical findings would potentially help in designing the experiments and aid in enhancing the selectivity and yield of vanillin.

Effects of calcium doping to oxygen reduction activity on Pr2-xCaxNiMnO6 cathode

Pr2-xCaxNiMnO6-δ (PCNMOx, x = 0.0–0.3) are prepared successfully by glycine-nitrate method. The effects of calcium doping to the crystal structure, the oxygen non-stoichiometry, and the cathode properties are evaluated by XRD, SEM, XPS and EIS. The double perovskite PCNMOx crystallize into a monoclinic structure with space group P21/n. The unit cell volumes and thermal expansion coefficients increase systematically with Ca2+ doping, due to the gradual generation of oxygen vacancies in the lattice. Pr2-xCaxNiMnO6-δ exhibits promising chemical compatibility with the electrolyte material Ce0.9Gd0.1O1.95 (GDC) at 1200 °C. The electrochemical characterization results indicate that both oxygen vacancy concentration and electrical conductivity play important roles to cathode properties. The optimum composition Pr1.8Ca0.2NiMnO6-δ shows the lowest polarization resistance of 0.18 Ω cm2 and highest peak power density of 0.3 W cm2 at 700 °C on GDC electrolyte supported fuel cell. The electrochemical impedance measurements under oxygen partial pressures, together with distribution of relaxation times analysis, identify three conjunctive elementary processes involved in the cathode reaction, and prove that the charge transfer process is the major rate-determining step of oxygen reduction reaction.

Elementary reaction modeling of reversible CO/CO2 electrochemical conversion on patterned nickel electrodes

CO/CO2 are the major gas reactant/product in the fuel electrode of reversible solid oxide cells (RSOC). This study proposes a two-charge-transfer-step mechanism to describe the reaction and transfer processes of CO-CO2 electrochemical conversion on a patterned Ni electrode of RSOC. An elementary reaction model is developed to couple two charge transfer reactions, C(Ni)+O2−(YSZ) ↔ CO(Ni)+(YSZ) +2e− and CO(Ni)+O2−(YSZ) ↔ CO2(Ni)+(YSZ)+2e−, with adsorption/desorption, surface chemical reactions and surface diffusion. This model well validates in both solid oxide electrolysis cell (SOEC) and solid oxide fuel cell (SOFC) modes by the experimental data from a patterned Ni electrode with 10 μm stripe width at different pCO (0–0.25 atm), pCO2 (0–0.35 atm) and operating temperature (600-700 °C). This model indicates SOEC mode is dominated by charge transfer step C(Ni)+O2−(YSZ)↔CO(Ni)+(YSZ) +2e−, while SOFC mode by CO(Ni)+ O2−(YSZ)↔CO2(Ni)+(YSZ)+2e− on the patterned Ni electrode. The sensitivity analysis shows charge transfer step is the major rate-determining step for RSOC, besides, surface diffusion of CO and CO2 as well as CO2 adsorption also plays a significant role in the electrochemical reaction of SOEC while surface diffusion of CO and CO2 desorption could be co-limiting in SOFC.

Investigation of Pr2NiMnO6‐Ce0.9Gd0.1O1.95 composite cathode for intermediate-temperature solid oxide fuel cells

The electrochemical performance of Pr2NiMnO6 (PNMO)-xCe0.9Gd0.1O1.95 (CGO) (x = 0–40 wt%) composite oxides as intermediate-temperature solid oxide fuel cell (IT-SOFC) cathode materials are evaluated. The electrochemical impedance spectroscopy (EIS) analysis results identify two consecutive electrode processes on the composite cathode. Among the various composites, PNMO-30CGO cathode exhibits the best electrochemical performance with the minimum polarization resistance of 0.23 Ω cm2 and the maximum exchange current density of 75 mA cm−2 at 700 °C in air. These values are almost constant even after 30-h operation. The oxygen reduction reaction (ORR) mechanism studies prove that the major rate-determining step is the charge-transfer process. Introducing CGO significantly improves the charge-transfer process, by increasing the triple phase boundary (TPB) length and oxygen vacancy concentration in the composite cathode.

Kinetic Comparisons of Heart and Kidney Na+,K+-ATPases

Most kinetic measurements of the partial reactions of Na+,K+-ATPase have been conducted on enzyme from mammalian kidney. Here we present a kinetic model that is based on the available equilibrium and kinetic parameters of purified kidney enzyme, and allows predictions of its steady-state turnover and pump current in intact cells as a function of ion and ATP concentrations and the membrane voltage. Using this model, we calculated the expected dependence of the pump current on voltage and extracellular Na+ concentration. The simulations indicate a lower voltage dependence at negative potentials of the kidney enzyme in comparison with heart muscle Na+,K+-ATPase, in agreement with experimental results. The voltage dependence is enhanced at high extracellular Na+ concentrations. This effect can be explained by a voltage-dependent depopulation of extracellular K+ ion binding sites on the E2P state and an increase in the proportion of enzyme in the E1P(Na+)3 state in the steady state. This causes a decrease in the effective rate constant for occlusion of K+ by the E2P state and hence a drop in turnover. Around a membrane potential of zero, negligible voltage dependence is observed because the voltage-independent E2(K+)2 → E1 + 2K+ transition is the major rate-determining step.

Kinetics of K+ Occlusion by the Phosphoenzyme of the Na+,K+-ATPase

Investigations of K+-occlusion by the phosphoenzyme of Na+,K+-ATPase from shark rectal gland and pig kidney by stopped-flow fluorimetry reveal major differences in the kinetics of the two enzymes. In the case of the pig enzyme, a single K+-occlusion step could be resolved with a rate constant of 342 (±26) s−1. However, in the case of the shark enzyme, two consecutive K+-occlusions were detected with rate constants of 391 (±19) s−1 and 48 (±2) s−1 at 24°C and pH 7.4. A conformational change of the phosphoenzyme associated with K+-occlusion is, thus, the major rate-determining step of the shark enzyme under saturating concentrations of all substrates, whereas for the pig enzyme the major rate-determining step under the same conditions is the E2 → E1 transition and its associated K+ deocclusion and release to the cytoplasm. The differences in rate constants of the K+ occlusion reactions of the two enzymes are paralleled by compensating changes to the rate constant for the E2 → E1 transition, which explains why the differences in the enzymes' kinetic behaviors have not previously been identified.

Dioxygen Activation for the Self-Degradation of Heme: Reaction Mechanism and Regulation of Heme Oxygenase

Heme oxygenase (HO) catalyzes the regiospecific conversion of heme to biliverdin, CO, and free iron through three successive oxygenation reactions. HO catalysis is unique in that all three O(2) activations are performed by the substrate itself. This Forum Article overviews our current understanding on the structural and biochemical properties of HO catalysis, especially its first and third oxygenation steps. The HO first step, regiospecific hydroxylation of the porphyrin alpha-meso-carbon atom, is of particular interest because of its sharp contrast to O(2) activation by cytochrome P450. HO was proposed to utilize the FeOOH species but not conventional ferryl hemes as a reactive intermediate for self-hydroxylation. We have succeeded in preparing and characterizing the FeOOH species of HO at low temperature, and our analyses of its reaction, together with mutational and crystallographic studies, reveal that protonation of FeOOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous autooxidation of the reactive alpha-meso-hydroxyheme in which the HO enzyme does not play a critical role. Further O(2) activation by verdoheme cleaves its porphyrin macrocycle to form biliverdin and free ferrous iron. This third step has been considered to be a major rate-determining step of HO catalysis to regulate the enzyme activity. Our reaction analysis strongly supports the FeOOH verdoheme as the key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, and the distal water is suggested to enhance the third step as expected from the similarity. The HO mechanistic studies highlight the catalytic importance of the distal hydrogen-bonding network, and this manuscript also involves our attempts to develop HO inhibitors targeting the unique distal structure.

Balancing Conformational and Oxidative Kinetic Traps during the Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) with Glutathione and Glutathione Disulfide

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.

Kinetic Resolution of a Tryptophan-radical Intermediate in the Reaction Cycle of Paracoccus denitrificans Cytochrome c Oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.

Kinetic Mechanism of Glutaryl-CoA Dehydrogenase

Glutaryl-CoA dehydrogenase (GCD) is a homotetrameric enzyme containing one noncovalently bound FAD per monomer that oxidatively decarboxylates glutaryl-CoA to crotonyl-CoA and CO2. GCD belongs to the family of acyl-CoA dehydrogenases that are evolutionarily conserved in their sequence, structure, and function. However, there are differences in the kinetic mechanisms among the different acyl-CoA dehydrogenases. One of the unanswered aspects is that of the rate-determining step in the steady-state turnover of GCD. In the present investigation, the major rate-determining step is identified to be the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD. Other steps are only partially rate-determining. This conclusion is based on the transit times of the individual reactions occurring in the active site of GCD.

NMR Structural Characterization and Computational Predictions of the Major Intermediate in Oxidative Folding of Leech Carboxypeptidase Inhibitor

The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.

Rate Limitation of the Na +,K +-ATPase Pump Cycle

The kinetics of Na +-dependent phosphorylation of the Na +,K +-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na +], and [Mg 2+], pH 7.4, and 24°C). The well-established effect of buffer composition on the E 2-E 1 equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E 2) yielded a 1/ τ value of 59 s −1. Addition of MgCl 2 (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/ τ to values of 129, 167, and 143 s −1, respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E 2 state to an E 1 or E 1-like state. The results thus demonstrate conclusively that the E 2 → E 1 transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E 2 → E 1 transition under physiological conditions could be estimated to be ∼65 s −1 for pig kidney enzyme and 90 s −1 for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (∼48 s −1 and ∼43 s −1 for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the α 1 isoform the E 2 → E 1 conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant.

Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase. The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined. The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme. The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme. For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step.

Stimulation of Open Complex Formation by Nicks and Apurinic Sites Suggests a Role for Nucleation of DNA Melting in Escherichia coli Promoter Function

We report the effects of depurination and prenicking at various positions of the phage lambda prmup-1Delta265 promoter DNA on the rate of open complex formation. We have found that depurination and prenicking at positions around the -10 region strongly stimulated the rate of open complex formation. Since nicking and depurination are known to destabilize DNA helical structure, our observations indicate that the instability of the -10 region is important for open complex formation. We further infer that (i) the nucleation of DNA melting, which occurs during the isomerization from the closed complex into the open complex, contributes to the rate of open complex formation; (ii) the nucleation of melting occurs around the -10 region; and (iii) the propagation of DNA melting from the nucleation region is not rate-limiting. In addition, we have found that depurination at several positions inhibited open complex formation. We used dimethyl sulfate modification protection studies to show that most of the guanine bases that are among these positions are in contact with RNA polymerase in the open complex.

Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2

Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case. The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds. Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.

29] Choline-phosphate cytidylyltransferase

Publisher Summary Choline-phosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase) catalyzes a major rate-determining step in the biosynthesis of phosphatidylcholine in mammalian cells. Both cytosolic and membrane fractions contain choline-phosphate cytidylyltransferase activity. Enzyme activity is determined by measuring the formation of radioactive CDPcholine from phospho[methyl- 14 C]choline. Two methods have been used to separate CDPcholine from phosphocholine: adsorption of CDPcholine by charcoal or separation of CDPcholine from phosphocholine by thin-layer chromatography. The charcoal adsorption method is rapid, sensitive, and reproducible. The ability to perform many assays (50–100) per day is an additional advantage. A variety of lipids have been used by investigators for the assay of cytidylyltransferase. It has been a common practice to include lipid in assays involving soluble forms of cytidylyltransferase. The molecular and kinetic properties of cytidylyltransferase are discussed this chapter.

Al<SUB>2</SUB>O<SUB>3</SUB>のN<SUB>2</SUB>気流中炭素熱還元によるAlNの生成反応

Experiments were made of the synthesis of AlN by reduction of Al2O3 with graphite powder in flowing N2 atmosphere at various temperatures between 1773 and 1973 K. At temperature below 1823 K, a temporary rise in the reaction rate was observed near the reaction degree of 50%. This discontinuous change was reflected in the residual carbon and AlN contents. At 1973 K, a small quantity of aluminum oxynitride was formed in the early stage of reaction. The reaction rate was hardly affected by the grain size of graphite, the pellet diameter and the flow rate of N2 gas. The examination of these results led to the conclusion that the diffusion of the reaction gas through the AlN layer formed around the Al2O3 grain is a major rate-determining step. Indeed, the reaction rate obeyed the rate equation which contains only the resistance of intraparticle diffusion.

A mathematical model of the penetration of polycyclic aromatic hydrocarbons through the bronchial lining layer

The enhanced carcinogenicity of particle-associated polycyclic aromatic hydrocarbons (PAHs) has previously been explained by their prolonged retention in the lung due to the slow release of these substances from their carrier particles. However, the mathematical model presented here indicates that the lipid-aqueous heterogenic structure of the bronchial lining layer (BLL) in itself may constitute the major rate-determining step in the overall transport of PAHs from carrier particles to the bronchial epithelium. Conditions within the BLL are then as important for the resulting cellular burdens of PAHs in the lung as are the properties of the carrier particles for these hydrocarbons. Thus, in addition to other vital functions in the respiratory tract, the lipids in lung surfactant can also be important in protecting the lung from the action of lipophilic carcinogens.

Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP +) by preparations of pure human liver MAO B : monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the K m,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, V max,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce V max,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.