Highly purified preparations of ATP:l-methionine S-adenosyltransferase (EC 188.8.131.52) of bakers' yeast display a virtually completely specific requirement for ATP. Kinetics analyses of the binding of a number of structural analogues and components of ATP have permitted assessment of the importance of various regions of the ATP molecular for "binding" and for "recognition" of the nucleotide. Adenosine or deoxyadenosine have almost no affinity for the enzyme, their mono- and dipphosphates are very weak inhibitors competitive with respect to ATP. Various nucleoside triphosphates are more powerful inhibitors competitive with respect to ATP and non-competitive with respect to l-methionine. Tripolyphosphate is an excellent inhibitor (Ki = 0.05 mM) and tetraphosphate also inhibits the enzyme (Ki = 0.18 mM). The following phosphonate analogues of ATP are powerful inhibitors: α,β-methylene-ATP (Ki = 0.55 mM); β,γ-methylene-ATP (Ki = 2.19 mM) and α,β-methylene-adenosine tetraphosphate (Ki = 0.17 mM). All of these phosphonates compete with ATP and the Ki values refer to this competition when l-methionine approaches saturation. It is concluded from kinetic studies with adenosine and its analogues, that the triphosphate moiety of ATP is of primary importance for the binding of the substrate to the enzyme, while the adenosyl group of ATP is of primary importance for the substrate recognition, but with low affinity for the enzyme. Kinetic patterns obtained with analogues of ATP as inhibitors are consistent with the previous proposition that the ATP:l-methionine S-adenosyltransferase reaction involves a random-ordered bi-mechanism. The results of the present studies also suggest that the adenosyl group remains enzyme-bound prior to its binding to the sulfur atom of l-methionine, and that the triphosphate site-directed recognition of the adenosyl moiety enables ATP to gain virtually absolute substrate specificity.
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