Abstract
Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.
Highlights
Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes
We report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by Cystathionine ␥-lyase (CSE) in vitro, is sensitive to preincubation of the enzyme with PPG
Our study reveals that CPC inhibits both the cystathionine and cysteine cleavage reactions catalyzed by CSE but spares the activity of the other enzymes involved in H2S synthesis (i.e. CBS and mercaptopyruvate sulfurtransferase (MST))
Summary
S-3-Carboxypropyl-L-cysteine inhibits cystathionine ␥-lyase–dependent hydrogen sulfide synthesis. We report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. We observed that CPC inhibited the ␥-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ؎ 3 and 180 ؎ 15 M, respectively. CSE belongs to the ␥-subfamily of PLP-dependent enzymes [16] and catalyzes the ␣-␥ elimination of cystathionine (formed in the CBS reaction) to cysteine, ␣-ketobutyrate, and ammonia (Fig. 1A). Our study reveals that CPC inhibits both the cystathionine and cysteine cleavage reactions catalyzed by CSE but spares the activity of the other enzymes involved in H2S synthesis (i.e. CBS and MST (and PLP-dependent CAT)). In cell lines and tissues like kidney, where cysteine concentrations are high, PPG is expected to be less effective at inhibiting the -elimination reaction of cysteine to form H2S
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