Structural Basis for the Inhibition Mechanism of Human Cystathionine γ-Lyase, an Enzyme Responsible for the Production of H2S

Abstract

Impairment of the formation or action of hydrogen sulfide (H 2 S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine ␤-synthase and cystathionine ␥-lyase (CSE) are two pyridoxal-5-phosphate (PLP)-dependent enzymes largely responsible for the production of H 2 S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP⅐PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSEmediated production of H 2 S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE⅐PLP⅐PAG complex highlights the particular importance of Tyr 114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

H2S Synthesizing Enzymes: Biochemistry and Molecular Aspects

“…Some of the commonly used pharmacological inhibitors of CSE are DL-propargylglycine (PAG) (Marcotte and Walsh 1975) which irreversibly inhibits CSE by physically obstructing the access of the substrate to the active site of the enzyme (Sun et al 2009) and the reversible inhibitor β-cyanoalanine (BCA) which has also been proposed as a competitive inhibitor of this enzyme (Pfeffer and Ressler 1967). However, these compounds have frequently been claimed to exhibit poor selectivity, require high concentrations and have limited ability to permeate the cell membrane (Szab o 2007).…”

Endogenous Production of H2S in the Gastrointestinal Tract: Still in Search of a Physiologic Function

“…The structure of CSE, bound to its inhibitor propargylglycine, has been recently solved ( Fig. 1) (28,61). This advance is likely to result in the development of new and more-selective enzyme inhibitors.…”

Hydrogen sulfide and inflammation: the good, the bad, the ugly and the promising

“…The addition of Na 2 S, NaSH or saturated solutions of H 2 S gas to aqueous solutions results in the instantaneous release of a bolus of H 2 S (and HS -), which dissipates in seconds [33,68]. It is highly unlikely that cells or tissues are ever exposed to H 2 S generated in such a rapid manner, generating high local concentrations of H 2 S, since endogenously produced H 2 S through CSE and CBS is relatively slow and sustained [68,92,109,110]. Furthermore, injection of these salts into animals results in a minimal increase in plasma H 2 S concentrations over a very short period of time [21,33,68,111].…”

“…While undoubtedly initially very useful pharmacological tools in a new but rapidly expanding research field, it is unlikely that these compounds are entirely specific since they target the pyridoxal phosphate (PLP) binding site of CSE and CBS [92] and other PLP-dependent and -independent enzymes (summarized in Table 1). This problem is exaggerated by the disparate concentration ranges commonly used in the laboratory (often used up to 10 mmol/l [93][94][95] …”