Solubilization and Characterization of the Initial Enzymes of the Dolichol Pathway from Yeast

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Abstract
Preparation and purification of substrate amounts of radioactive as well as non‐radioactive dolichyl diphosphate N‐acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I–III). The test conditions are described in detail.All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35‐fold and 70‐fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+.The Km values for UDP‐N‐acetylglucosamine for enzyme I and II were 15 and 10 μM, respectively, and for GDP‐mannose (enzyme III) 7 μM. The apparent Km values for the lipophilic acceptor was 180 μM for enzyme I (dolichyl phosphate), 40 μM for enzyme II (dolichyl diphosphate N‐acetylglucosamine) and 17 μM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol × h−1× mg protein−1. All reactions were inhibited by nucleoside diphosphates.