Enzymatic analysis of ?-ketoglutaramate--a biomarker for hyperammonemia.
ABSTRACT: Two enzymatic assays were developed for the analysis of ?-ketoglutaramate (KGM)-an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to ?-ketoglutarate (KTG) via a reaction catalyzed by ?-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (?=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids.
Project description:Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to ?-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to ?-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ?-amidase. Transamination of glutamine results in formation of the corresponding ?-keto acid, namely, ?-ketoglutaramate (KGM). KGM is hydrolyzed by ?-amidase to ?-ketoglutarate and ammonia. The net glutaminase II reaction is: L?-?Glutamine?+???-?keto acid?+?H2O ? ??-?ketoglutarate?+?L?-?amino acid?+?ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.
Project description:?-Ketoglutaramate (KGM) is the ?-keto acid analogue of glutamine, which exists mostly in equilibrium with a lactam form (2-hydroxy-5-oxoproline) under physiological conditions. KGM was identified in human urine and its concentration quantified by gas chromatography/mass spectrometry (GC/MS). The keto acid was shown to be markedly elevated in urine obtained from patients with primary hyperammonemia due to an inherited metabolic defect in any one of the five enzymes of the urea cycle. Increased urinary KGM was also noted in other patients with primary hyperammonemia, including three patients with a defect resulting in lysinuric protein intolerance and one of two patients with a defect in the ornithine transporter I. These findings indicate disturbances in nitrogen metabolism, most probably at the level of glutamine metabolism in primary hyperammonemia diseases. Urinary KGM levels, however, were not well correlated with secondary hyperammonemia in patients with propionic acidemia or methylmalonic acidemia, possibly as a result, in part, of decreased glutamine levels. In conclusion, the GC/MS procedure has the required lower limit of quantification for analysis of urinary KGM, which is markedly increased in urea cycle disorders and other primary hyperammonemic diseases.
Project description:The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25 °C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.
Project description:Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.
Project description:Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ?-amidase. hNit2/?-amidase plays a crucial metabolic role by catalyzing the hydrolysis of ?-ketoglutaramate (the ?-keto analog of glutamine) and ?-ketosuccinamate (the ?-keto analog of asparagine), yielding ?-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and ?-keto-?-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/?-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/?-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/?-amidase was determined with ?-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/?-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/?-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.
Project description:BACKGROUND:Citrin, encoded by SLC25A13, is a component of the malate-aspartate shuttle, which is the main NADH-transporting system in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), which usually resolves within the first year of life. However, small numbers of adults with citrin deficiency develop hyperammonemic encephalopathy, adult-onset type II citrullinemia (CTLN2), which leads to death due to cerebral edema. Liver transplantation is the only definitive therapy for patients with CTLN2. We previously reported that a lactose (galactose)-restricted and medium-chain triglyceride (MCT)-supplemented formula is notably effective for patients with NICCD. Citrin deficiency may impair the glycolysis in hepatocytes because of an increase in the cytosolic NADH/NAD+ ratio, leading to an energy shortage. MCT administration can provide energy to hepatocytes and was expected to have a good effect on CTLN2. METHODS:An MCT supplementation therapy under a low-carbohydrate formula was administered to five patients with CTLN2. Four of the patients had episodes of hyperammonemic encephalopathy, and one patient had postprandial hyperammonemia with no symptoms. RESULTS:One of the patients displaying hyperammonemic encephalopathy completely recovered with all normal laboratory findings. Others notably improved in terms of clinical and or laboratory findings with no hyperammonemic symptoms; however, the patients displayed persistent mild citrullinemia and occasionally had postprandial mild hyperammonemia most likely due to an irreversible change in the liver. CONCLUSIONS:An MCT supplement can provide energy to hepatocytes and promote hepatic lipogenesis, leading to a reduction in the cytosolic NADH/NAD+ ratio. MCT supplementation under a low-carbohydrate formula could be a promising therapy for CTLN2 and should also be used to prevent CTLN2 to avoid irreversible liver damage.
Project description:NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD(+), suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD(+) dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.
Project description:Type-II NADH:quinone oxidoreductases (NDH-2s) are an important element of microbial pathogen electron transport chains and an attractive drug target. Despite being widely studied, its mechanism and catalysis are still poorly understood in a hydrophobic membrane environment. A recent report for the Escherichia coli NDH-2 showed NADH oxidation in a solution-based assay but apparently showed the reverse reaction in electrochemical studies, calling into question the validity of the electrochemical approach. Here we report electrochemical catalysis in the well-studied NDH-2 from Caldalkalibacillus thermarum (CthNDH-2). In agreement with previous reports, we demonstrated CthNDH-2 NADH oxidation in a solution assay and electrochemical assays revealed a system artifact in the absence of quinone that was absent in a membrane system. However, in the presence of either immobilized quinone or mobile quinone in a membrane, NADH oxidation was observed as in solution-phase assays. This conclusively establishes surface-based electrochemistry as a viable approach for interrogating electron transfer chain drug targets.
Project description:Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.
Project description:Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.