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

Abstract

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 β-lyase). Recombinant human BCAT_m and BCAT_c were shown to have β-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 β-chloro-l-alanine. Human BCAT_m is a much more effective β-chloro-l-alanine β-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 β-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl-l-cysteine]. Benzothiazolyl-l-cysteine, however, inhibits the l-leucine–α-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT_m. In the presence of β-lyase substrates and α-ketoisocaproate (the α-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the β position, β-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the β-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is ∼170–280 for BCAT_m and ∼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.

Introduction

PLP-dependent enzymes have been classified into four families with different fold types. Most aminotransferases, including the well-studied AspAT [1], [2], belong to fold type I [3]. On the other hand, BCAT_m and the cytosolic isoform (BCAT_c) belong to the fold type IV family of PLP-dependent enzymes [3], [4], [5]. Only two other enzymes are currently known to belong to the fold type IV family. These are bacterial DAAT [6], [7], [8], [9] and ADCL [10], [11]. ADCL catalyzes the conversion of 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate (a β-lyase reaction).

BCAT_m is widely expressed in tissues including kidney and brain (reviewed in Ref. [12]). On the other hand, BCAT_c is present only in nervous tissue, and to a lesser extent in ovary and placenta [12]. BCAT_m has a well-defined role in the metabolism of whole-body branched-chain amino acids [13]. The metabolic role of BCAT_c is less clear. The occurrence of both isoforms in nervous tissue, however, suggests a unique aspect to branched-chain amino acid metabolism in brain. Indeed, the enzymes may be important in replenishing the nitrogen of neurotransmitter glutamate, and in nitrogen cycling between astrocytes and neurons [14], [15], [16], [17], [18], [19], [20], [21], [22], [23].

It has long been known that aminotransferases such as DAAT [24], cytAspAT [25], [26], [27], mitAspAT [26], and AlaAT [28] can catalyze β-lyase reactions with amino acids containing a good leaving group in the β position. Aminoacrylate [CH₂C(NH₃⁺)CO₂⁻] is released from the active site, which then undergoes bond rearrangement and hydrolysis to pyruvate and ammonia. The net reaction (Eq. (1)) is:

$$\text{XCH}{}_2\text{CH}\text{(}\text{CO}{}_2{}^{\mathrm{-}}\text{)}\text{NH}{}_3{}^{\mathrm{+}}\text{+}\text{H}{}_2\text{O}\text{→}\text{CH}{}_3\text{C}\text{(}\text{O}\text{)}\text{CO}{}_2{}^{\mathrm{-}}\text{+}\text{NH}{}_4{}^{\mathrm{+}}\text{+}\text{X}{}^{\mathrm{-}}$$

The early studies used β-chloro-l-alanine (or the d-isomer) and l-serine O-sulfate [24], [25], [26], [27], [28]. DAAT, cytAspAT, mitAspAT, and AlaAT are all inactivated syncatalytically during turnover of the β-lyase substrates [24], [25], [26], [27], [28]. More recently, it was shown that cytAspAT [29], [30], [31], [32], [33], AlaAT [30], [33], and mitAspAT [34] can catalyze β-lyase reactions with cysteine S-conjugates containing a good leaving group in the β position (Eq. (1), X=RS). Slow syncatalytic inactivation of purified pig heart cytAspAT, pig heart AlaAT, and rat liver mitAspAT was shown to occur with DCVC and TFEC [33], [34].

Several halogenated alkenes (e.g. trichloroethylene, tetrachloroethylene, and tetrafluoroethylene) are heavily used in industry. In the case of trichloroethylene, there is some concern not only for the exposed workers, but for the general population, because this compound is a major environmental contaminant. Trichloroethylene causes renal and liver tumors in experimental animals (e.g. Refs. [35], [36]). Although there has been some past debate on the issue, trichloroethylene is almost certainly a human renal carcinogen (e.g. Refs. [37], [38], [39]). Tetrafluoroethylene, the precursor of Teflon™, produces both hepatocellular carcinomas and kidney cell adenomas in rodents [40], and chronic inhalation of this haloalkene results in damage to the renal proximal tubules in rats [41]. Lifetime exposure to tetrachloroethylene (perchloroethylene, perc) induces a low level of renal tumors in rats [42]. Halogenated alkenes are metabolized at least in part to the corresponding cysteine S-conjugates (DCVC, TFEC, and CTFC are the cysteine S-conjugates corresponding to trichloroethylene, tetrafluoroethylene, and chlorotrifluororethylene, respectively.) Much evidence suggests that cysteine S-conjugates are a major factor in the nephrotoxicity of halogenated alkenes (e.g. Ref. [43] and references cited therein). Within the kidney, the proximal tubules, especially the S3 region, are especially sensitive. Toxicity of DCVC has been demonstrated in isolated rat (e.g. Ref. [44]) and human [45] kidney proximal tubules, and in cultured human proximal tubule cells [46]. Toxicity is due in part to the high reactivity of the sulfur-containing fragment eliminated by the action of cysteine S-conjugate β-lyases. Evidence suggests that the fragments eliminated from DCVC and TFEC (and CTFC) breakdown to a thioketene [47] and a dihalothionoacetyl fluoride [43], [48], respectively, both of which act as thioacylating agents particularly of lysine residues in proteins [49], [50], [51], [52]. Proteins in the kidney mitochondria are especially vulnerable to thioacylation after rats are administered TFEC. Several mitochondrial enzymes of energy metabolism are inactivated in kidney cells [53], [54], [55], PC12 cells [56], and hepatocytes [55] exposed to TFEC. Because of the potential for human exposure to halogenated alkenes in the workplace and in the environment, it is important to characterize the cysteine S-conjugate β-lyases that may contribute to the bioactivation of halogenated cysteine S-conjugates. (For reviews, see Refs. [57], [58], [59], [60], [61], [62], [63].)

Inasmuch as (a) β-lyase activity appears to be a general property of many aminotransferases including DAAT (a fold class IV PLP enzyme), and (b) ADCL (another fold class IV enzyme) naturally catalyzes a β-lyase reaction, the fold class IV BCAT isozymes should, theoretically, also be able to catalyze effective β-lyase reactions. The present work shows that both BCAT isozymes catalyze β-lyase reactions with toxic halogenated cysteine S-conjugates and with β-chloro-l-alanine. Turnover was shown to lead to inactivation. The relatively bulky BTC was found to be a β-lyase substrate and inactivator of BCAT_m. Inactivation was more pronounced at higher pH values. BTC was neither a β-lyase substrate nor an irreversible inhibitor of BCAT_c. On the other hand, BTC inhibited transamination between leucine and α-ketoglutarate catalyzed by both enzymes, but inhibition was somewhat more pronounced with BCAT_m.