Studies on the kinetics and stoichiometry of inactivation of Pseudomonas omega-amino acid:pyruvate transaminase by gabaculine.

Burnett, George H.; Yonaha, Kazuo; Toyama, Seizen; Soda, Kenji; Walsh, Christopher T.

doi:10.1016/s0021-9258(19)86191-4

Cited by 27 publications

(2 citation statements)

References 17 publications

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“…On the basis of this proposed model, only one subunit of the enzyme would be active at a time. The occurrence of a negative cooperative behavior (flipflop mechanism) of dimers or tetramers of other PLP enzymes has been proposed on the basis of either their patterns of inhibition in the presence of suicide inhibitors (42) or activity levels upon PLP titration of reconstituted enzymes (43). Negative cooperative behavior of PLP enzymes provides a mechanistic reason for the conservation of dimeric and tetrameric organization among enzymes of this family.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

2010

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Cysteine is the major sulfur donor for thio cofactors in bacterial and eukaryotic systems. The first step in sulfur mobilization involves a PLP-dependent enzymatic mechanism. During catalysis, free cysteine is converted into alanine with the concomitant formation of a persulfide bond with the catalytic cysteine residue, thus forming a covalent enzyme intermediate. Cysteine desulfurases in their persulfurated forms serve as donors at the intersection of various cellular sulfur-requiring pathways. Most Gram-positive bacteria, including Bacillus subtilis, contain a cysteine desulfurase gene sufS located adjacent to the gene encoding the proposed Fe-S cluster scaffold SufU. In this work, we identified the participation of SufU as a substrate in the SufS catalytic mechanism. Development of a sensitive method for detection of alanine formed in the SufS reaction enabled the identification of its associated mechanistic features. Steady-state kinetic analysis of alanine formation provided evidence of a double-displacement mechanism (ping-pong) of the cysteine:SufU sulfurtransferase reaction catalyzed by SufS. Results from site-directed mutagenesis of the catalytic cysteine (SufS(C361A)) and iodoacetamide alkylation of SufU support the occurrence of persulfide sulfur transfer steps in the mechanism of SufS.

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Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

2010

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“…This minimizes the nonspecific modification of other cellular components, which may result in side effects and toxicity. Thus, epoxy-ATP joins a select group of "perfect" inhibitors including gabaculine (inactivates GABA transaminase, partition ratio = 1; Burnett et al, 1980), vinylglycine (inactivates L-aspartate transaminase, partition ratio = 1; Rando, 1974), and isatoic anhydride (inactivates chymotrypsin, partition ratio = 1; Moorman & Abeles, 1982).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor

Catalano¹,

Benkovic²

1989

Biochemistry

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The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.

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Proteinreaktive Naturstoffe

2005

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In der Post‐Genom‐Ära steht man vor der gewaltigen Aufgabe, die Strukturen und Funktionen zehntausender codierter Proteine zuzuweisen. Zur Analyse der Proteinfunktion werden neue Technologien mit großer Bandbreite entwickelt, etwa die aktivitätsbasierte Proteinanalyse (activity‐based protein profiling, ABPP). Bei diesem Verfahren sollen auf das aktive Zentrum gerichtete chemische Sonden entwickelt werden, um die Enzyme in ganzen Proteomen zu analysieren. Derartige chemische Proteomtechnologien können von proteinreaktiven Naturstoffen lernen, die Enzyme der unterschiedlichsten Klassen durch ein bemerkenswert vielfältiges Spektrum an Mechanismen kovalent modifizieren und somit eine reiche Quelle an Konzepten für die Entwicklung gegen aktive Zentren gerichteter Proteomsonden bilden. Hier werden wir anhand unterschiedlicher Naturstoffe zeigen, wie ihre Wirkmechanismen zum Fortschritt chemischer Proteomtechnologien wie ABPP beigetragen haben.

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Studies on the kinetics and stoichiometry of inactivation of Pseudomonas omega-amino acid:pyruvate transaminase by gabaculine.

Cited by 27 publications

References 17 publications

Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor

Proteinreaktive Naturstoffe

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