Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its alpha,gamma-elimination.
The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH3SeH from seleno-l-methionine (l-SeMet) by the alpha,gamma-elimination reaction. The l-SeMet alpha,gamma-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The Km value of the enzyme for the catalysis of l-SeMet was 15.5 mM, and the V<I->max was 0.29 units/mg protein. Pyridoxal 5'-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50 degrees C; it catalyzed the alpha,gamma-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the alpha,beta-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inert. Therefore, the purified enzyme was different from the bacterial l-methionine gamma-lyase that metabolizes l-SeMet to CH3SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the alpha,gamma-elimination reaction of l-SeMet and immediately converts it to CH3SeH, an important metabolite of Se.
Okuno
Tomofumi
T
Division of Environmental Health, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan.
Motobayashi
Shinji
S
Ueno
Hitoshi
H
Nakamuro
Katsuhiko
K
D000375:Aging; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000302:isolation & purification*; D002384:Catalysis; D002845:Chromatography; D005260:Female; D006710:Homocysteine / Q000737:chemistry; D006714:Homoserine / Q000737:chemistry; D006801:Humans; D007420:Intestine, Large / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D000432:Methanol / Q000031:analogs & derivatives* / Q000737:chemistry; D008715:Methionine / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D009132:Muscles / Q000378:metabolism; D016566:Organoselenium Compounds / Q000737:chemistry*; D012643:Selenium / Q000737:chemistry; D012645:Selenomethionine / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature
D016566:Organoselenium Compounds; D006710:Homocysteine; C019003:methaneselenol; D006714:Homoserine; D012645:Selenomethionine; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C505901:SeMet alpha,gamma-elimination enzyme, mouse; C021558:L-methionine gamma-lyase; D012643:Selenium; D000645:Ammonium Sulfate; D000432:Methanol