Title | Transition-state structure of human 5'-methylthioadenosine phosphorylase. |
Publication Type | Journal Article |
Year of Publication | 2006 |
Authors | Singh V, Schramm VL |
Journal | J Am Chem Soc |
Volume | 128 |
Issue | 45 |
Pagination | 14691-6 |
Date Published | 2006 Nov 15 |
ISSN | 0002-7863 |
Keywords | Crystallography, X-Ray, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Conformation, Purine-Nucleoside 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. Intrinsic KIEs were obtained for [1'-(3)H], [1'-(14)C], [2'-(3)H], [4'-(3)H], [5'-(3)H(2)], [9-(15)N], and [Me-(3)H(3)] MTAs. The primary intrinsic KIEs (1'-(14)C and 9-(15)N) suggest that MTAP has a dissociative S(N)1 transition state with its 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 for the adenine leaving group, whereas the alpha-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 Angstrom 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 degrees. The [Me-(3)H(3)] and [5'-(3)H(2)] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulfur with the sigma (C-H) antibonding orbitals. Human MTAP is characterized by a late S(N)1 transition state with significant participation of the phosphate nucleophile. |
DOI | 10.1021/ja065419p |
Alternate Journal | J Am Chem Soc |
PubMed ID | 17090056 |
PubMed Central ID | PMC2522318 |
Grant List | R01 GM041916 / GM / NIGMS NIH HHS / United States R01 GM041916-20 / GM / NIGMS NIH HHS / United States R37 GM041916 / GM / NIGMS NIH HHS / United States GM 41916 / GM / NIGMS NIH HHS / United States |
Transition-state structure of human 5'-methylthioadenosine phosphorylase.
Submitted by Vipender Singh on