Synthesis and characterization of selenolipoylated H-protein of the glycine cleavage system.
H-protein of the glycine cleavage system has a lipoic acid prosthetic group. Selenolipoic acid is a lipoic acid analog in which both sulfur atoms are replaced by selenium atoms. Two isoforms of bovine lipoyltransferase that are responsible for the attachment of lipoic acid to H-protein had an affinity for selenolipoyl-AMP and transferred the selenolipoyl moiety to bovine apoH-protein comparable to lipoyl-AMP. Selenolipoylated H-protein was overexpressed in Escherichia coli and purified. Selenolipoylated H-protein was 26% as effective as lipoylated H-protein in the glycine decarboxylation reaction, in which reduction of the diselenide bond of selenolipoylated H-protein is catalyzed by P-protein. The diselenide form of selenolipoylated H-protein was a poor substrate for L-protein, and the rate of reduction was 0.5% of that of lipoylated H-protein. The rate of the overall glycine cleavage reaction with selenolipoylated H-protein was <1% of that with lipoylated H-protein. These results are consistent with the difference in the redox potential between the diselenide and disulfide bonds. In contrast, selenolipoylated H-protein showed three times as high glycine-14CO2 exchange activity as lipoylated H-protein, presumably because the rate of reoxidation of reduced selenolipoylated H-protein is much higher than that of lipoylated H-protein.
Fujiwara
K
K
Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan.
Okamura-Ikeda
K
K
Packer
L
L
Motokawa
Y
Y
D000217:Acyltransferases / Q000378:metabolism; D000594:Amino Acid Oxidoreductases; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli; D005998:Glycine / Q000378:metabolism*; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D007700:Kinetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D011994:Recombinant Proteins; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D000217:Acyltransferases; C077290:H-protein lipoyltransferase; D012643:Selenium; D005998:Glycine