Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5′-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1′-3H, 1′-14C, 2′-3H, 4′-3H, 5′-3H, 9-15N, and Me-3H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1′-3H and small 1′-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 Å from the anomeric carbon. The small β-secondary 2′-3H KIE of 1.044 corresponds to a modest 3′-endo conformation for ribose and a H1′-C1′- C2′-H2′ dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4′-3H KIE is due to hyperconjugation between the lone pair (np) of O3′ and the antibonding (σ*) orbital of the C4′-H4′ group, and the methyl-3H3 KIE is due to hyperconjugation between the np of sulfur and the σ* of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.
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