Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae.
L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.
Yamagata
Shuzo
S
Department of Biotechnology, Faculty of Agriculture, Gifu University, Gifu 501-1193.
Yasugahira
Tomoko
T
Okuda
Yuriko
Y
Iwama
Tomonori
T
D002384:Catalysis; D003542:Cystathionine gamma-Lyase / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013053:Spectrophotometry; D013844:Thiazoles / Q000737:chemistry; D013997:Time Factors
D010710:Phosphates; D017680:Potassium Compounds; D013844:Thiazoles; C013216:potassium phosphate; D003542:Cystathionine gamma-Lyase; D003545:Cysteine