Transition-State Structure of Human 5‘-Methylthioadenosine Phosphorylase

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Abstract
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 SN1 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 orbitals. Human MTAP is characterized by a late SN1 transition state with significant participation of the phosphate nucleophile.

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