Measurement of Cysteine S‐Conjugate β‐Lyase Activity

Abstract

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH 2 CH(NH 3 + )CO 2 − ] and selenium Se-conjugates[RSeCH 2 CH(NH 3 + )CO 2 − ] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a β-lyase reaction, but catalyze a non-physiological β-lyase side reaction that depends on the electron-withdrawing properties of the -SR or -SeR moiety. In the case of the cysteine S-conjugates, if the eliminated RSH is stable the compound may be S-thiomethylated and excreted (thiomethyl shunt) or Sglucuronidated and harmlessly excreted [the possibility that RSeH compounds may be similarly metabolized has not been extensively studied]. If, however, RSH is chemically reactive the cysteine S-conjugate may be toxic as a result of the β-lyase reaction. The cysteine S-conjugate β-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. Keywords Ammonium; cysteine S-conjugates; cysteine S-conjugate β-lyases; S-(1,2-dichlorovinyl)-Lcysteine; S-(1,1,2,2-tetrafluoroethyl)-L-cysteine; pyruvateMany potentially toxic electrophiles, whether of endogenous origin (e.g. α,β-unsaturated aldehydes derived from the oxidation of lipids) or of exogenous origin (e.g. some drugs), are detoxified through the mercapturate pathway (Figure 1). In addition, some non-electrophilic toxins may be converted to electrophiles that are then detoxified through the mercapturate pathway. The electrophile is sequentially converted to the glutathione S-conjugate, cysteinylglycine S-conjugate, cysteinyl S-conjugate and N-acetyl cysteine S-conjugate (mercapturate) by the action of glutathione S-transferases, γ-glutamyltransferase (γ-glutamyltranspeptidase), dipeptidases and N-acetyl transferases, respectively. The mercapturate is generally more hydrophilic and water soluble than is the starting electrophile and is readily excreted in the urine or bile.If the cysteine S-conjugate contains a good leaving group (nucleofuge) in the β position the metabolism of the electrophile may be diverted irreversibly away from the mercapturate by the action of cysteine S-conjugate β-lyases. These PLP-containing enzymes catalyze the conversion of the cysteine S-conjugate [RSCH 2 CH(NH 3 + )CO 2 − ] to RSH and aminoacrylate

Tetrachlorethen [MAK Value Documentation in German language, 2017]

“…Diese Enzyme sind in Geweben von Säugetieren weit verbreitet. So wird bei Ratten die GTK in fast allen untersuchten Geweben nachgewiesen (Cooper et al 2010;Cooper und Pinto 2006).…”

A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1

“…Other inhibitors, such as BFF-122 [33], showed 1.5-fold increased activity rather than inhibiting the enzyme, in the β-elimination assay, but not in the coupled or transamination assay. α-ketoglutarate [34] did not show any inhibitory or inducing activity towards the KYAT1 enzyme reaction in either the transamination nor β-elimination assay (data not shown); α-ketoglutarate is known to increase the KYAT1 activity [17]. Indole-3-pyruvic acid [34,35] showed a 50% reduced activity in the β-elimination activity assay and 20% reduced activity in transamination activity assay; we could not observe these effects in the coupled assay because the color of this compound interferes with the assay.…”

“…KYAT1 β-elimination activity was measured with pure protein or whole-cell extract according to the published procedure [17]. The assay protocol was adjusted to measure pure protein activity in 96-well plate readers for users' convenience.…”

Synthesis of enantiopure 18F-trifluoromethyl cysteine as a structure-mimetic amino acid tracer for glioma imaging

“…Even though both 2L and 2D were stable in vitro , there was a marked difference in stability between the two enantiomers in vivo . One reasonable explanation is that 2L might serve as a preferential substrate for cysteine S -conjugate β-lyases and underwent an enzyme-catalyzed β-elimination reaction 67, 68. Structurally, 2D is also a cysteine S -conjugate but showed relatively good in vivo stability, presumably because the β-elimination reaction proceeded with high L-stereoselectivity.…”