Interaction of Zn2؉ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn 2؉ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 ؎ 0.02 M. Activation is a consequence of Zn 2؉ binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn 2؉ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn 2؉ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.

SummaryCysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously . Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-M r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

Low (C 1a2 = 1.5U U10 37

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-L-selenocysteine. L-3-(2-Naphthyl)alanine, L-3-(1-naphthyl)alanine, 5-S-L-cysteinyldopamine and 5-S-L-cysteinyl-L-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/Lamino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTKcatalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-L-cysteinyldopamine, but not with 5-S-L-cysteinyl-L-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-L-cysteinyldopamine, 5-S-L-cysteinyl-L-DOPA and Se-methyl-L-selenocysteine in human tissues and their biological relevance are discussed. KeywordsCysteine S-conjugate β-lyase; 5-S-L-cysteinyl-L-DOPA; 5-S-L-cysteinyldopamine; glutamine transaminase K; Se-methyl-L-selenocysteine Glutamine transaminase K (GTK) 1 purified from rat and bovine tissues accepts a large number of neutral, aromatic and sulfur/selenium-containing substrates [1][2][3][4][5][6]. GTK catalyzes *Corresponding author: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA. Fax: +1 914 594 4058, E-mail address: arthur_cooper@nymc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Abbreviations used: BTC, benzothiazolyl-L-cysteine; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DOPA, dihydroxyphenylalanine; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, glutathione; KAT, kynurenine aminotransferase; KGDHC, α-ketoglutarate dehydrogenase complex; KGM, α-ketoglutaramate; KMB, α-keto-γ-methiolbutyrate; MBTH, 3-methyl-2-benzothiazolinone hydrazone; MDH, malate dehydrogenase; MPA, metaphosphoric acid; PDHC, pyruvate dehydrogenase complex; PLP, pyridoxal 5′-phosphate; rhGTK, recombinant human glutamine transaminase K; TFEC, S- (1,1,2,2-tetrafluoroethyl) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transamination of kynurenine to kynurenate, ...

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.

Eight active transglutaminases (TGs) (TGs 1-7 and factor XIIIa) are expressed in mammals, of which TGs 1-3 (Kim et al. 1999) 1 and 6 (Hadjivassilou et al. 2008) are present in human brain. The major reaction thus far attributed to the cerebral TGs is transamidation. In this reaction the carboxamide moiety of a Q residue [-C(O)NH 2 ] is converted to a substituted carboxamide [-C(O)NHR] by nucleophilic attack of an amine [RNH 2 ] such as various mono-, di-, and polyamines or the e amino group of a K residue (Lorand and Graham 2003). Of the possible transamidation linkages, theisopeptide linkage formed between Q and K resides, is the most commonly studied. GGEL bonds occur both within and between polypeptide chains, and thereby contribute to the formation of stable soluble and insoluble polymers. TGs also cross-link proteins via bis-c-glutamylpolyamine bridges between Q residues (Piacentini et al. 1988). These linkages are formed by two successive transamidations: the first utilizes a free polyamine to generate a c-glutamylpolyamine residue, which becomes the amine-bearing substrate for a second transamidation. bis-c-Glutamylpolyamine cross-links are formed at least as frequently as those involving GGEL -dependent enzymes that catalyze a variety of modifications of glutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a number of amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to either regulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity is thought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclear palsy. Strategies designed to interfere with TG activity have some benefit in animal models of Huntington and Parkinson diseases. The following review summarizes the involvement of TGs in neurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agents in these diseases.

Many cancer cells have a strong requirement for glutamine. As an aid for understanding this phenomenon the 18F-labeled 2S,4R stereoisomer of 4-fluoroglutamine [(2S,4R)4-FGln] was previously developed for in vivo positron emission tomography (PET). In the present work, comparative enzymological studies of unlabeled (2S,4R)4-FGln and its deamidated product (2S,4R)4-FGlu were conducted as an adjunct to these PET studies. Our findings are as follows: Rat kidney preparations catalyze the deamidation of (2S,4R)4-FGln. (2S,4R)4-FGln and (2S,4R)4-FGlu are substrates of various aminotransferases. (2S,4R)4-FGlu is a substrate of glutamate dehydrogenase, but not of sheep brain glutamine synthetase. The compound is, however, a strong inhibitor of this enzyme. Rat liver cytosolic fractions catalyze a γ-elimination reaction with (2S,4R)4-FGlu, generating α-ketoglutarate. Coupling of a deamidase reaction with this γ- elimination reaction provides an explanation for the previous detection of 18F− in tumors exposed to [18F](2S,4R)4-FGln. One enzyme contributing to this reaction was identified as alanine aminotransferase, which catalyzes competing γ-elimination and aminotransferase reactions with (2S,4R)4-FGlu. This appears to be the first description of an aminotransferase catalyzing a γ-elimination reaction. The present results demonstrate that (2S,4R)4-FGln and (2S,4R)4-FGlu are useful analogues for comparative studies of various glutamine- and glutamate-utilizing enzymes in normal and cancerous mammalian tissues, and suggest that tumors may metabolize (2S,4R)4-FGln in a generally similar fashion to glutamine. In plants, yeast and bacteria a major route for ammonia assimilation involves the consecutive action of glutamate synthase plus glutamine synthetase (glutamate synthase cycle). It is suggested that (2S,4R)4-FGln and (2S,4R)4-FGlu will be useful probes in studies of ammonia assimilation by the glutamate synthase pathway in these organisms. Finally, glutamine transaminases are conserved in mammals, plants and bacteria, and probably serve to close the methionine salvage pathway, thus linking nitrogen metabolism to sulfur metabolism and one-carbon metabolism. It is suggested that (2S,4R)4-FGln may be useful in studies of the methionine salvage pathway in a variety of organisms.

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine fi cysteamine fi hypotaurine fi taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (£ 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wildtype littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.