PURIFICATION AND PROPERTIES OF AN ENZYME INVOLVED IN THE ATP-DEPENDENT ACTIVATION OF THE METHANOL-2-MERCAPTOETHANESULFONIC ACID METHYLTRANSFERASE REACTION IN METHANOSARCINA-BARKERI

In Methanosarcina barkeri the transfer of the methyl group from methan ol to 2-mercaptoethanesulfonic acid is catalyzed by the concerted acti on of two methyltransferases. The first one is the corrinoid-containin g methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT(1)), which binds the methyl group of methanol to its corrinoid prosthetic g roup. MT(1) is only catalytically active when the cobalt atom of the c orrinoid is present in the highly reduced Co(I) state, In the course o f its purification and even during catalysis, MT(1) becomes oxidativel y inactivated. The enzyme, however, may be reductively reactivated by a suitable reducing system (hydrogen and hydrogenase), ATP, and an enz yme called methyltransferase activation protein (MAP). In order to elu cidate its role in the reactivation process, MAP was purified to appar ent homogeneity. The protein had an M(r) = 60,000. Preincubation of th e enzymic components involved with 8-azido-ATP or with ATP demonstrate d MAP to be the primary site of action of ATP. In agreement herewith, the protein was auto phosphorylated by [gamma-P-32]ATP in a 1:1 stoich iometry, Phosphorylated MAP substituted for ATP in the activation of M T(1), and the addition of increasing amounts of MAP phosphate resulted in a corresponding increase of active MT(1). However, in the presence of limiting amounts of MAP, maximal activation of MT(1) could be achi eved during a lag phase provided ATP was present, indicating that MAP acts as a catalyst. This paper is the first to report on the presence, isolation, and function of a phosphorylated protein in a methanogenic archaeon.