Solubilization and properties of polyprenyl phosphate: GDP- d-mannose mannosyl transferase

A soluble enzyme which catalyzes the formation of dolichyl β- d-mannosyl phosphate has been prepared from encysting cultures of Acanthamoeba castellanii. The enzyme is relatively specific for GDP- d-mannose in that GDP- d-glucose and various uridine nucleotides do not serve as substrates. Uridine diphosphate d-glucose is not an inhibitor at 100-fold molar excess concentration, but GDP- d-glucose, GDP, and GMP do inhibit the reaction at relatively high concentrations. The apparent K m for GDP- d-mannose is approximately 0.25 μ m and that for dolichyl phosphate is approximately 3.3 μ m. The enzyme has a pH optimum of 7.0, a temperature optimum of 27 °C, and requires a divalent cation. Magnesium, cobalt, and manganese salts will serve as cofactors but maximum activity is produced by Mn 2+. No loss of activity is evident after storage for 2 weeks at −70 °C, but half the activity was lost within 3 days at 0 °C, and a third of the activity was lost within 2 weeks at −20 °C.