Transition State Structure of 5‘-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli and Its Similarity to Transition State Analogues

Abstract

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'-(3)H, 1'-(14)C, 2'-(3)H, 4'-(3)H, 5'-(3)H, 9-(15)N, and Me-(3)H(3) 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'-(3)H and small 1'-(14)C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (D(N)A(N)) (S(N)1) 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 A from the anomeric carbon. The small beta-secondary 2'-(3)H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'-H2' dihedral angle of 53 degrees at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-(3)H KIE is due to hyperconjugation between the lone pair (n(p)) of O3' and the antibonding (sigma) orbital of the C4'-H4' group, and the methyl-(3)H(3) KIE is due to hyperconjugation between the n(p) of sulfur and the sigma 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.

5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni

“…A kinetic isotope effect analysis showed that the enzyme utilizes a highly dissociative mechanism with a transition state that has considerable oxocarbenium ion character (Fig. 3) (28). Mutagenesis and structural studies have shown that there are three conserved residues that are absolutely essential for the activity of this enzyme: Glu-12, Glu-174, and .…”

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

“…Alkaline phosphatase was from Roche. Ribokinase and phosphoribosyl-α-1-pyrophosphate synthetase were prepared as previously described (36,37). Hypoxanthine-guanine phophoribosyltransferase was a kind gift from Keith Hazleton of this laboratory.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Studies utilizing kinetic isotope effects (KIEs) and crystal structures of MTANs in complex with known transition state analogues have allowed for transition state modeling of MTANs from E. coli, S. pneumoniae, Neisseria meningitidis, S. aureus, and V. cholerae and the subsequent design of analogue inhibitors (120)(121)(122)(149)(150)(151)(152)(153). Thus far, it appears that most MTANs share a fully dissociated S N 1 transition state characterized by a ribooxacarbenium ion (121,122,149,152), with the exception of MTANs from (149).…”

“…Thus far, it appears that most MTANs share a fully dissociated S N 1 transition state characterized by a ribooxacarbenium ion (121,122,149,152), with the exception of MTANs from (149). The majority of the transition state analogue inhibitor characterization has been performed using purified MTANs in vitro.…”