Human methionine S-adenosyltransferase (MAT2A) catalyzes the formation of S-adenosylmethionine (SAM) from ATP and methionine. Synthetic lethal genetic analysis has identified MAT2A as' an anticancer target in tumor cells lacking expression of S'-methylthioadenosine phosphorylase (MTAP). Approximately 15% of human cancers are MTAP(-/-). The remainder can be rendered MTAP(-) through MTAP inhibitors. We used kinetic isotope effect (KIE), commitment factor.(c), and binding isotope effect (BIE) measurements combined with quantum mechanical (QM) calculations to solve the transition state structure of human MAT2A. The reaction is charactetized by an advanced SN2 transition state. The bond foxming from the nudeophilic methionine suffnr to the 5'-C of ATP is 2.03 angstrom at the transition state (bond order of 0.67). Departure of the leaVing group triphosphate of ATP is well advanced and forms a 2.32 angstrom bond between the 5'-C of ATP and the oxygen of the triphosphate.(bond order of 0.23)..Interaction of MAT2A with its MAT2B regulatory subunit causes no change in the intrinsic KIEs, indicating the same transition gate structure. The transition state for MAT2A is' more advanced along the reaction coordinate (more product-like) than that from the near-symmetrical transition state of methionine adenosylttansferase from E. coli.