Membrane-associated thiamin triphosphatase. II. Activation by divalent cations.
Activation of membrane-associated thiamin triphosphatase from rat brain requires a divalent cation (Mg2+, Ca2+, or Mn2+). The optimum concentration of Mg2+ necessary for maximal enzyme activity varies with substrate concentration; conversely, the maximal rate of hydrolysis attainbale by increasing thiamin triphosphate concentration is directly proportional to [Mg2+] for all levels of Mg2+ below that of the substrate. Under appropriate conditions, the Km of the thiamin triphosphatase for Mg2+ and for thiamin triphosphate are shown to be identical. Dissociation constants (Kd) for the binding of Mg2+ to thiamin triphosphate, thiamin diphosphate, and thiamin were determined; kinetic data re-expressed in terms of [Mg2+-thiamin triphosphate] conform to simple single substrate predictions, suggesting that the true enzyme substrate may be the Mg2+-thiamin triphosphate complex. Excess free Mg2+ inhibits thiamin triphosphatase activity competitively while excess free thiamin triphosphate in concentrations up to 10 times Km has no effect on the membrane-bound enzyme.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives
D008345:Manganese; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D013835:Thiamine Pyrophosphate; D002118:Calcium