Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma.

BACKGROUND: The mitochondrial branched-chain aminotransferase (BCATm) is a recently discovered cancer marker with a poorly defined role in tumour progression. METHODS: To understand how a loss of function of BCATm affects cancer, the global knockout mouse BCATmKO was challenged with EL-4 lymphoma under different diet compositions with varying amounts of branched-chain amino acids (BCAAs). Next, the growth and metabolism of EL-4 cells were studied in the presence of different leucine concentrations in the growth medium. RESULTS: BCATmKO mice experienced delayed tumour growth when fed standard rodent chow or a normal BCAA diet. Tumour suppression correlated with 37.6- and 18.9-fold increases in plasma and tumour BCAAs, 37.5% and 30.4% decreases in tumour glutamine and alanine, and a 3.5-fold increase in the phosphorylation of tumour AMPK in BCATmKO mice on standard rodent chow. Similar results were obtained with a normal but not with a choice BCAA diet. CONCLUSIONS: Global deletion of BCATm caused a dramatic build-up of BCAAs, which could not be utilised for energy or amino acid synthesis, ultimately delaying the growth of lymphoma tumours. Furthermore, physiological, but not high, leucine concentrations promoted the growth of EL-4 cells. BCATm and BCAA metabolism were identified as attractive targets for anti-lymphoma therapy.

Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein.

Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 µg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.

BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1.

Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

S-Nitrosoglutathione inactivation of the mitochondrial and cytosolic BCAT proteins: S-nitrosation and S-thiolation.

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Functional impact of high protein intake on healthy elderly people.

Decline in muscle mass, protein synthesis, and mitochondrial function occurs with age, and amino acids are reported to enhance both muscle protein synthesis and mitochondrial function. It is unclear whether increasing dietary protein intake corrects postabsorptive muscle changes in aging. We determined whether a 10-day diet of high [HP; 3.0 g protein x kg fat-free mass (FFM)(-1) x day(-1)] vs. usual protein intake (UP; 1.5 g protein x kg FFM(-1) x day(-1)) favorably affects mitochondrial function, protein metabolism, and nitrogen balance or adversely affects insulin sensitivity and glomerular filtration rate (GFR) in 10 healthy younger (24+/-1 yr) and 9 older (70+/-2 yr) participants in a randomized crossover study. Net daily nitrogen balance increased equally in young and older participants, but postabsorptive catabolic state also increased, as indicated by higher whole body protein turnover and leucine oxidation with no change in protein synthesis. Maximal muscle mitochondrial ATP production rate was lower in older people, with no change occurring in diet. GFR was lower in older people, and response to HP was significantly different between the two groups, with a significant increase occurring only in younger people, thus widening the differences in GFR between the young and older participants. In conclusion, a short-term high-protein diet increased net daily nitrogen balance but increased the postabsorptive use of protein as a fuel. HP did not enhance protein synthesis or muscle mitochondrial function in either young or older participants. Additionally, widening differences in GFR between young and older patients is a potential cause of concern in using HP diet in older people.

Regulatory control of human cytosolic branched-chain aminotransferase by oxidation and S-glutathionylation and its interactions with redox sensitive neuronal proteins.

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.

Redox regulation and trapping sulfenic acid in the peroxide-sensitive human mitochondrial branched chain aminotransferase.

The human branched chain aminotransferase enzymes are key regulators of glutamate metabolism in the brain and are among a growing number of redox-sensitive proteins. Studies that use thiol-specific reagents and electrospray ionization mass spectrometry demonstrate that the mitochondrial BCAT enzyme has a redox-active CXXC center, which on oxidation forms a disulfide bond (RSSR), via a cysteine sulfenic acid intermediate. Mechanistic details of this redox regulation were revealed by the use of mass spectrometry and dimedone modification. We discovered that the thiol group at position C315 of the CXXC motif acts a redox sensor, whereas the thiol group at position C318 permits reversible regulation by forming an intrasubunit disulphide bond. Because of their roles in redox regulation and catalysis, there is a growing interest in cysteine sulphenic acids. Therefore, development of chemical tags/methods to trap these transient intermediates is of immense importance.

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism.

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat.

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0+/-0.02, 1.3+/-0.1 and 2.1+/-0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.

Engineering of a recombinant vitamin K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic acid forming capacity: evidence for a functional CXXC redox center in the system.

The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.

Neuroglial metabolism in the awake rat brain: CO2 fixation increases with brain activity.

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n = 23) were infused simultaneously with 14C-bicarbonate and [1-13C]glucose for up to 1 hr. The 14C and 13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and 13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic 13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the 13C-labeling time courses alone and with 14C confirmed significant glial TCA cycle activity (V(PDH)((g)), approximately 0.5 micromol x gm(-1) x min(-1)) relative to the glutamate-glutamine cycle (V(NT)) (approximately 0.5-0.6 micromol x gm(-1) x min(-1)). The glial TCA cycle rate was approximately 30% of total TCA cycle activity. A high pyruvate carboxylase rate (V(PC), approximately 0.14-0.18 micromol x gm(-1) x min(-1)) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.

Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme.

The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by site-directed mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H(2)O(2)) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in k(cat) with no significant change in K(m). The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced k(cat) (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H(2)O(2). The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H(2)O(2) resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H(2)O(2)-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the "resolving cysteine", allowing for reversible formation of a disulfide bond.

Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves.

We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme.

Human mitochondrial branched chain aminotransferase: structural basis for substrate specificity and role of redox active cysteines.

Crystal structures of the fold type IV pyridoxal phosphate (PLP)-dependent human mitochondrial branched chain aminotransferase (hBCATm) reaction intermediates have provided a structural explanation for the kinetically determined substrate specificity of hBCATm. The isoleucine side chain in the ketimine intermediate occupies a hydrophobic binding pocket that can be defined by three surfaces. Modeling of amino acids on the ketimine structure shows that the side chains of nonsubstrate amino acids such as the aromatic amino acids, alanine, or aspartate either are unable to interact through van der Waals' interactions or have steric clashes. The structural and biochemical basis for the sensitivity of the mammalian BCAT to reducing agents has also been elucidated. Two cysteine residues in hBCATm, Cys315 and Cys318 (CXXC), are part of a redox-controlled mechanism that can regulate hBCATm activity. The residues surrounding Cys315 and Cys318 show considerable sequence conservation in the prokaryotic and eukaryotic BCAT sequences, however, the CXXC motif is found only in the mammalian proteins. The results suggest that the BCAT enzymes may join the list of enzymes that can be regulated by redox status.

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate beta-lyases, but turnover leads to inactivation.

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT(m) and BCAT(c)) are homodimers in the fold type IV class of pyridoxal 5'-phosphate-containing enzymes that also contains D-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a beta-lyase). Recombinant human BCAT(m) and BCAT(c) were shown to have beta-lyase activity toward three toxic cysteine S-conjugates [S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine] and toward beta-chloro-L-alanine. Human BCAT(m) is a much more effective beta-chloro-L-alanine beta-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT(m), but not BCAT(c), also exhibits measurable beta-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl-L-cysteine]. Benzothiazolyl-L-cysteine, however, inhibits the L-leucine-alpha-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT(m). In the presence of beta-lyase substrates and alpha-ketoisocaproate (the alpha-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the beta position, beta-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the beta-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is approximately 170-280 for BCAT(m) and approximately 40-50 for BCAT(c). Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.

Crystal structures of human mitochondrial branched chain aminotransferase reaction intermediates: ketimine and pyridoxamine phosphate forms.

The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution. The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme. The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis. A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place. Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance. As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions. The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor. These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin. Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT. Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond. This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase.

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.