Transition-state structure of human 5′-methylthioadenosine phosphorylase

Kinetic isotope effects (KIEs) and computer modeling using density functional theory were used to approximate the transition state of human 5′-methylthioadenosine phosphorylase (MTAP). KIEs were measured on the arsenolysis of 5′-methylthioadenosine (MTA) catalyzed by MTAP and were corrected for the forward commitment to catalysis. Intrinsic KIEs were obtained for [1′-³H], [1′-¹⁴C], [2′- ³H], [4′-³H], [5′-³H₂], [9-¹⁵N], and [Me-³H₃] MTAs. The primary intrinsic KIEs (1′-¹⁴C and 9-¹⁵N) suggest that MTAP has a dissociative S_N1 transition state with its cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-¹⁵N intrinsic KIE of 1.039 also establishes an anionic character for the adenine leaving group, whereas the α-primary 1′- ¹⁴C KIE of 1.031 indicates significant nucleophilic participation at the transition state. Computational matching of the calculated EIEs to the intrinsic isotope effects places the oxygen nucleophile 2.0 Å from the anomeric carbon. The 4′-³H KIE is sensitive to the polarization of the 3′-OH group. Calculations suggest that a 4′-³H KIE of 1.047 is consistent with ionization of the 3′-OH group, indicating formation of a zwitterion at the transition state. The transition state has cationic character at the anomeric carbon and is anionic at the 3′-OH oxygen, with an anionic leaving group. The isotope effects predicted a 3′-endo conformation for the ribosyl zwitterion, corresponding to a H1′-C1′-C2′-H2′ torsional angle of 33°. The [Me-³H₃] and [5′-³H₂] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulfur with the σ* (C-H) antibonding orbitale. Human MTAP is characterized by a late S_N1 transition state with significant participation of the phosphate nucleophile.