Transition-State Structure of Human 5‘-Methylthioadenosine Phosphorylase

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. MTAs. The primary intrinsic KIEs (1′-14 C and 9-15 N) suggest that MTAP has a dissociative S N 1 transition state with cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-15 N intrinsic KIE of 1.039 also establishes an anionic character to the adenine leaving group, whereas the α-primary 1′-14 C 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′-3 H KIE is sensitive to the polarization of the 3′-OH group. Calculations suggest that a 4′-3 H 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-3 H 3 ] and [5′-3 H] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulphur with the σ* (C-H) antibonding orbitals. Human MTAP is characterized by a late, S N 1 transition state with significant participation of the phosphate nucleophile.

Transition states of native and drug-resistant HIV-1 protease are the same

“…Both computational methods give similar results; however, 15 N KIEs derived from B3LYP calculations are smaller than expected for most structures (near unity). This observation is consistent with previous studies in the literature that show B1LYP and B3LYP underpredict 15 N KIEs (39)(40)(41). Furthermore, the M06-2X functional gives more accurate results than B3LYP in bond breaking and forming reactions (42).…”

Enzymatic Transition State Theory and Transition State Analogue Design

“…SAM is a critical metabolite both as a precursor for polyamine synthesis and for methylation reactions that are essential to provide epigenetic control through methylation of histones and CpG islands in DNA. Kinetic isotope effects for arsenolysis of MTA by MTAP indicated full loss of the N-ribosidic bond and significant nucleophilic participation of the arsenate (33).…”

Molecular Determinants of Substrate Specificity in Plant 5′-Methylthioadenosine Nucleosidases

“…Kinetic isotope effect studies have established this alternative reaction pathway in Streptococcus pneumonia MTAN and human MTAP. 29,30 The crystal structures of S. pneumonia MTAN (PDB code 1ZOS) and human MTAP (PDB code 1K27) demonstrate that, like AtMTAN1, water is not excluded from the active sites of these enzymes upon ligand binding.…”