Abstract
Although there is a growing recognition of the significance of hydrogen sulfide (H(2)S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H(2)S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine beta-synthase and cystathionine gamma-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H(2)S from cysteine and homocysteine. The gamma-replacement reaction, which condenses two molecules of homocysteine, yields H(2)S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a beta-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the alpha,beta-elimination of cysteine accounts for approximately 70% of H(2)S generation. However, the relative importance of homocysteine-derived H(2)S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 microm), the alpha,gamma-elimination and gamma-replacement reactions of homocysteine together are predicted to account for approximately 90% of H(2)S generation by CSE. Excessive H(2)S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.
Highlights
H2S is the newest member of a growing list of gaseous signaling molecules that modulate physiological functions [1,2,3]
The absorption spectrum of purified cystathionine ␥-lyase (CSE) is typical of a pyridoxal phosphate (PLP)-dependent enzyme with a maximum at 428 nm and a 280:428 nm ratio of ϳ1:6 (Fig. 2)
Despite the growing interest in H2S biology and the therapeutic potential of H2S-releasing compounds [2], surprisingly little is known about the enzymatic production of this gas and how it may be influenced by changes in sulfur amino acid levels in disease states
Summary
Recombinant human CSE (polymorphic variant S403) was expressed in the Escherichia coli strain BL21(DE3) using an expression plasmid generously provided by Dr Marcus Wahl (Max Planck Institute, Mantinsried, Germany). After the reaction mixture (980 l) containing 100 mM Hepes buffer (pH 7.4), 0.4 mM lead acetate, and varying concentrations of substrate (homocysteine, cysteine, or both) was preincubated at 37 °C for 4 min, 20 g of CSE was added to the assay mixture to initiate the reaction, which was monitored at 37 °C for 3 min. 1 ml of the assay mixture containing 100 mM Hepes buffer (pH 7.4) and varying concentrations of homocysteine or cysteine was preincubated for 5 min at 37 °C, and the reaction was initiated by adding 20–50 g of CSE. The reaction mixture (1-ml final volume) contained 100 mM Hepes buffer (pH 7.4), 0.4 mM lead acetate, 5 M cystathionine, 100 M cysteine, and either 10, 40, or 200 M homocysteine. Control reaction mixtures from which CSE was omitted were run separately
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