l-norleucine on high glucose-induced insulin sensitivity and mitochondrial function in skeletal muscle cells.

l-norleucine, an isomer of leucine, stimulates the anabolic process of insulin. However, it is not known if and how it improves insulin sensitivity and insulin resistance. This experiment describes the generation of an insulin resistance model using high glucose-induced cells and the administration of 1.0 mmol/L l-norleucine for 48 h, to observe the effects on metabolism and gene expression in skeletal muscle cells. The results showed that l-norleucine significantly increased mitochondrial ATP content, decreased the amount of reactive oxygen species (ROS) and promoted the expression of mitochondrial generation-related genes TFAM, AMPK, PGC-1α in cells under high glucose treatment; at the same time, l-norleucine also increased glucose uptake, suggesting that l-norleucine increased insulin sensitivity and improved insulin resistance. This study suggesting that l-norleucine improves insulin resistance by ameliorating oxidative stress damage of mitochondria, improving mitochondrial function, and improving insulin sensitivity in skeletal muscle cell caused by high glucose, rather than by altering mitochondrial efficiency.

Degradation of the Indospicine Toxin from Indigofera spicata by a Mixed Population of Rumen Bacteria.

The leguminous plant species, Indigofera linnaei and Indigofera spicata are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with Indigofera spicata plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC-MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the Indigofera plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of Indigofera plant material by grazing ruminant livestock.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Pyrraline Formation Modulated by Sodium Chloride and Controlled by Encapsulation with Different Coating Materials in the Maillard Reaction.

Advanced glycation end products (AGEs), which are present in heat-processed foods, have been associated with several chronic diseases. Sodium chloride (NaCl) modulates the formation of furfurals and acrylamide in the Maillard reaction; however, the effects of NaCl on AGE formation are inconsistent. In this study, we investigated the effects of NaCl on pyrraline formation using glucose-lysine model systems. NaCl, especially at 0.50%, promoted Maillard browning and pyrraline formation, with a simultaneous increase in the 3-deoxyglucosone concentration. To reduce the rate of pyrraline formation, NaCl coated with different gums and starches were used. The results showed that NaCl encapsulation is an effective approach to mitigate pyrraline and 3-deoxyglucosone formation. The content of NaCl in the microparticles were 284 ± 12, 269 ± 6, 258 ± 8, 247 ± 10, 273 ± 16, and 288 ± 15 mg/g (coated with waxy maize starch, normal maize starch, HYLON VII high amylose maize starch, gelatinized resistant starch, xanthan gum, and gum arabic, respectively). The heat resistance of the coating material was negatively correlated with the pyrraline and 3-deoxyglucosone formation, whereas the solubility of the coating material had the opposite results. Coating the material with gum had little effects on the reduction of pyrraline and 3-deoxyglucosone.

Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo.

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).

Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis.

Studies of heterochronic parabiosis demonstrated that with age, the composition of the circulatory milieu changes in ways that broadly inhibit tissue regenerative capacity. In addition, local tissue niches have age-specific influences on their resident stem cells. Here we use bio-orthogonal proteome labeling for detecting in vivo proteins present only in transplanted myoblasts, but not in host tissue, and proteins exclusive to one young mouse and transferred during parabiosis to its old partner. We use a transgenic mouse strain that ubiquitously expresses a modified tRNA methionine synthase, metRS, which preferentially incorporates the methionine surrogate azido-nor-leucine (ANL) into newly generated proteins. Using click chemistry and a modified antibody array to detect ANL-labeled proteins, we identify several 'young' systemic factors in old regenerating muscle of the heterochronic parabiotic partners. Our approach enables the selective profiling of mammalian proteomes in mixed biological environments such as cell and tissue transplantation, apheresis or parabiosis.Clarifying the source of proteins in mixed biological environments, such as after transplantation or parabiosis, remains a challenge. Here, the authors address this need with a mouse strain that incorporates a methionine derivate into proteins, allowing for their detection using click chemistry and antibody arrays.

In Vitro Biodegradation of Hepatotoxic Indospicine in Indigofera spicata and Its Degradation Derivatives by Camel Foregut and Cattle Rumen Fluids.

The known accumulation of the hepatotoxin indospicine in tissues of camels and cattle grazing Indigofera pasture plants is unusual in that free amino acids would normally be expected to be degraded during the fermentation processes in these foregut fermenters. In this study, in vitro experiments were carried out to examine the degradability of indospicine of Indigofera spicata by camel and cattle foregut microbiota. In the first experiment, a 48 h in vitro incubation was carried out using foregut fluid samples that were collected from 15 feral camels and also a fistulated cow. Degradability of indospicine ranged between 97% and 99%, with the higher value of 99% for camels. A pooled sample of foregut fluids from three camels that were on a roughage diet was used in a second experiment to examine the time-dependent degradation of indospicine present in the plant materials. Results indicated that camels' foregut fluids have the ability to biodegrade ∼99% of the indospicine in I. spicata within 48 h of incubation and produced 2-aminopimelamic acid and 2-aminopimelic acid. The time-dependent degradation analysis showed rapid indospicine degradation (65 nmol/h) during the first 8-18 h of incubation followed by a slower degradation rate (12 nmol/h) between 18 and 48 h. Indospicine degradation products were also degraded toward the end of the experiment. The results of these in vitro degradation studies suggest that dietary indospicine may undergo extensive degradation in the foregut of the camel, resulting in trace levels after 48 h. The retention time for plant material in the camel foregut varies depending on feed quality, and the results of this study together with the observed accumulation of indospicine in camel tissues suggest that, although indospicine can be degraded by foregut fermentation, this degradation is not complete before the passage of the digesta into the intestine.

Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l-norleucine.

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Transformation of Free and Dipeptide-Bound Glycated Amino Acids by Two Strains of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae transforms branched-chain and aromatic amino acids into higher alcohols in the Ehrlich pathway. During microbiological culturing and industrial fermentations, this yeast is confronted with amino acids modified by reducing sugars in the Maillard reaction (glycation). In order to gain some preliminary insight into the physiological "handling" of glycated amino acids by yeasts, individual Maillard reaction products (MRPs: fructosyllysine, carboxymethyllysine, pyrraline, formyline, maltosine, methylglyoxal-derived hydroimidazolone) were administered to two strains of S. cerevisiae in a rich medium. Only formyline was converted into the corresponding α-hydroxy acid, to a small extent (10 %). Dipeptide-bound pyrraline and maltosine were removed from the medium with concomitant emergence of several metabolites. Pyrraline was mainly converted into the corresponding Ehrlich alcohol (20-60 %) and maltosine into the corresponding α-hydroxy acid (40-60 %). Five specific metabolites of glycated amino acids were synthesized and characterized. We show for the first time that S. cerevisiae can use glycated amino acids as a nitrogen source and transform them into new metabolites, provided that the substances can be transported across the cell membrane.

Accumulation, Persistence, and Effects of Indospicine Residues in Camels Fed Indigofera Plant.

Indospicine (l-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as Indigofera linnaei and Indigofera spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs, and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution, and histopathological effects of dietary indospicine on camels. Six young camels (2-4 years old), weighing 270-390 kg, were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam-flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day, designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy, and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw), followed by the muscle, heart, and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining three camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated monoexponential decreases.

Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates.

Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs) in foods. Peptide-enriched drinks (PEDs) are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr). In this study we determined free-form pyrraline (Free-Pyr) and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH), soy protein hydrolysate (SPH) and collagen protein hydrolysate (CPH). A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE). The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD) of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent) of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent) of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

High-level biosynthesis of norleucine in E. coli for the economic labeling of proteins.

The residue-specific labeling of proteins with non-canonical amino acids (ncAA) is well established in shake flask cultures. A key aspect for the transfer of the methodology to larger scales for biotechnological applications is the cost of the supplemented ncAAs. Therefore, we established a scalable bioprocess using an engineered host strain for the biosynthesis of the methionine analog norleucine at titers appropriate for the efficient and economic labeling of proteins. To enhance the biosynthesis of norleucine, which is a side-product of the branched chain amino acid pathway, we deleted all three acetolactate synthase isoforms of the methionine auxotrophic Escherichia coli expression strain B834(DE3). Additionally, we overexpressed leuABCD to boost the biosynthesis of norleucine. We systematically analyzed the production of norleucine under the conditions for its residue-specific incorporation in bioreactor cultures that had a 30-fold higher cell density than shake flask cultures. Under optimized conditions, 5g/L norleucine was biosynthesized. This titer is two times higher than the standard supplementation with norleucine of a culture with comparable cell density. We expect that our metabolically engineered strain for the improved biosynthesis of norleucine in combination with the proposed bioprocess will facilitate the efficient residue-specific labeling of proteins at a reasonable price in scales beyond the shake flask.

Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Stability of Individual Maillard Reaction Products in the Presence of the Human Colonic Microbiota.

Maillard reaction products (MRPs) are taken up in substantial amounts with the daily diet, but the majority are not transported across the intestinal epithelium. The aim of this study was to obtain first insights into the stability of dietary MRPs in the presence of the intestinal microbiota. Four individual MRPs, namely, N-ε-fructosyllysine (FL), N-ε-carboxymethyllysine (CML), pyrraline (PYR), and maltosine (MAL), were anaerobically incubated with fecal suspensions from eight human volunteers at 37 °C for up to 72 h. The stability of the MRPs was measured by HPLC with UV and MS/MS detections. The Amadori product FL could no longer be detected after 4 h of incubation. Marked interindividual differences were observed for CML metabolism: Depending on the individual, at least 40.7 ± 1.5% of CML was degraded after 24 h of incubation, and the subjects could thus be tentatively grouped into fast and slow metabolizers of this compound. PYR was degraded by 20.3 ± 4.4% during 24 h by all subjects. The concentration of MAL was not significantly lowered in the presence of fecal suspensions. In no case could metabolites be identified and quantified by different mass spectrometric techniques. This is the first study showing that the human colonic microbiota is able to degrade selected glycated amino acids and possibly use them as a source of energy, carbon, and/or nitrogen.

Investigation into the misincorporation of norleucine into a recombinant protein vaccine candidate.

A high level of norleucine misincorporation was detected in a recombinant methionine-rich protein vaccine candidate expressed in E. coli K12. An investigation was conducted to evaluate a simple remediation strategy to reduce norleucine misincorporation and to determine if the phenomenon was either (a) due to the depletion of methionine during fermentation, (b) a result of the cultivation environment, or (c) a strain-specific effect. While supplementation with exogenous methionine improved product quality, the undesirable biosynthesis of non-standard amino acids such as norleucine and norvaline persisted. In contrast, non-standard amino acid biosynthesis was quickly minimized upon selection of an appropriate fed-batch process control strategy, fermentation medium, and nutrient feed. By expressing the same protein in E. coli BL21(DE3), it was determined that the biosynthesis of norleucine and norvaline, and the misincorporation of norleucine into the protein were primarily attributed to the use of E. coli K12 as the host for protein expression.

New method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside the host cell.

UNLABELLED: Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRS(NLL)) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed "click" reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRS(NLL)-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRS(NLL)-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRS(NLL)-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS. IMPORTANCE: Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp.

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli.

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes.

Determination of hepatotoxic indospicine in Australian camel meat by ultra-performance liquid chromatography-tandem mass spectrometry.

Indospicine is a hepatotoxic amino acid found in Indigofera plant spp. and is unusual in that it is not incorporated into protein but accumulates as the free amino acid in the tissues (including muscle) of animals consuming these plants. Dogs are particularly sensitive to indospicine, and secondary poisoning of dogs has occurred from the ingestion of indospicine-contaminated horse meat and more recently camel meat. In central Australia, feral camels are known to consume native Indigofera species, but the prevalence of indospicine residues in their tissues has not previously been investigated. In this study, a method was developed and validated with the use of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine the level of indospicine in camel meat samples using isotopically labeled indospicine as an internal standard. UPLC-MS/MS analysis showed that the method is reproducible, with high recovery efficiency and a quantitation limit of 0.1 mg/kg. Camel meat samples from the Simpson Desert were largely contaminated (≈50%) by indospicine with levels up to 3.73 mg/kg (fresh weight) determined. However, the majority of samples (95%) contained less than 1 mg/kg indospicine.

The specificity and kinetic mechanism of branched-chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis.

UNLABELLED: Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation. DATABASE: Branched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2.