Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cy steine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochio metric pyridoxal phosphate. The enzyme was not functional as an L-alanine t ransaminase, and had a strong preference for L-cysteine over D-cysteine. Cy stalysin preferred small alpha-L-amino acids as substrates or inhibitors an d was far more active towards L-cysteine than towards the other standard am ino acids that undergo pyridoxal phosphate-dependent beta-elimination react ions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated sma ll modifications to the carboxylate of L-cysteine (i.e., the methyl and eth yl esters of L-cysteine were good substrates), but the smallest possible pe ptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor subs trate. These results, combined with the implicit requirement for a free ami ne for pyridoxal phosphate-dependent reactions, imply that cystalysin canno t catabolize cysteine residues located within peptides. Cystalysin has Mich aelis-Menten kinetics towards L-cysteine, and there was little or no inhibi tion by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated wi th H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein agg regates. Erythrocytes retained the ability to reduce methemoglobin in the p resence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalan ine was the substrate, indicating an absolute requirement for H2S productio n. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.