Highly purified preparations of ATP:L-methionine S-adenosyltransferase (EC 126.96.36.199) have been obtained from autolysates of bakers' yeast. Kinetic studies using a sensitive radioactive assay reveal considerable deviation from MichaelisMenten kinetics characterized by downward inflexions in double-reciprocal plots of initial velocity with respect to either L-methionine or ATP concentration. When product formation is reduced by lowering the temperature or by shortening the incubation time, so that the net accumulation of endogenous S-adenosyl-L-methionine (AMet) becomes negligible, a presteady-state lag period can be clearly demonstrated. This delay in product formation is shortened by addition of low levels of S-adenosyl-L-methionine and prolonged by addition of tripolyphosphate. Initial velocity studies and dead-end inhibition studies suggest a random order of addition of substrates. As the reaction proceeds, the rate-limiting step shifts from hydrolysis of tripolyphosphate to the formation of AMet. S-Adenosyl-L-methionine displays stimulatory and inhibitory effects (at low and high concentrations, respectively) on both its own synthesis and tripolyphosphate hydrolysis. Consequently, the enzyme displays biphasic regulatory control by AMet in addition to product inhibition. It is proposed that the release of S-adenosyl-L-methionine from the product site results in the inhibition of the overall reaction by virtue of the formation of an enzyme-tripolyphosphate complex in which tripolyphosphate is either very slowly or not at all hydrolyzed. In the presence of endogenous S-adenosyl-L-methionine the hydrolysis of tripolyphosphate by the enzyme is activated in an “hysteretic” manner and is followed by the release of all products. The conformational L-methionine analogs, L-2-amino-4-hexynoic acid, DL-2-amino-/rai-4-hexenoic acid, S-tri-fluoromethyl-L-homocysteine, and 1-aminocyclopentanecar-boxylic acid, are competitive inhibitors with respect to L-methionine and noncompetitive with respect to ATP, whereas GTP is a competitive inhibitor with respect to ATP and noncompetitive with respect to L-methionine.
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