Involvement of thiol groups in the reaction mechanism of Mn(2+)-activated alkaline p-nitrophenylphosphate phosphatase of the extreme halophilic archaebacterium Halobacterium halobium.
Halobacterium halobium contains a cytosolic alkaline p-nitrophenylphosphate phosphatase which is selectively activated by Mn2+ ions (Bonet et al., 1991: Int. J. Biochem. 12, 1445-1451). We investigated the reaction mechanism of the enzyme in the presence and the absence of Mn2+ by means of pH studies and the application of some group-specific reagents. A pKes 8.4 of a group in the Mn(2+)-enzyme-substrate complex involved in catalysis could not be detected when catalysis in the absence of the cation was analysed. Only the enzyme with bound Mn2+ was inhibited by p-hydroxymercuribenzoate. Reducing agents stimulated the Mn(2+)-dependent activity but had no effect on the native enzyme activity. Our results indicate that the reaction mechanism is different whether the activating cation is present or not, and that the reaction mechanism in the presence of Mn2+ involves thiol groups.
Bonet
M L
ML
División de Bioquímica, Facultad de Ciencias, Universidad de Alicante, Spain.
Llorca
F I
FI
Cadenas
E
E
D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019617:Halobacterium salinarum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008623:Mercaptoethanol / Q000494:pharmacology; D011190:Potassium Cyanide / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology
D002413:Cations, Divalent; D006902:Hydroxymercuribenzoates; D013438:Sulfhydryl Compounds; C030601:4-hydroxymercuribenzoate; D008345:Manganese; D008623:Mercaptoethanol; D009597:4-Nitrophenylphosphatase; D003545:Cysteine; D011190:Potassium Cyanide; D004229:Dithiothreitol