Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus.
L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.
Grabowski
R
R
Laboratorium für Mikrobiologie, Philipps Universität Marburg, Federal Republic of Germany.
Buckel
W
W
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010462:Peptostreptococcus / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013455:Sulfur / Q000032:analysis*
D011732:Pyridoxal Phosphate; D013455:Sulfur; D007501:Iron; D012695:L-Serine Dehydratase