Catalytic Strategy of S-Adenosyl-l-homocysteine Hydrolase:  Transition-State Stabilization and the Avoidance of Abortive Reactions

Abstract

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containing the intermediate analogue neplanocin A with the analogue bound in its 3'-keto form at the active sites of all of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzyme is in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs. Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F. L. (1991) J. Biol. Chem. 266, 21616-21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem. 267, 3205-3213. Porter, D. J. (1998) J. Biol. Chem. 268, 66-73] suggests elements of the catalytic strategy of AdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that in the absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrate and ceases motion. Probably concurrently with oxidation of the substrate to its 3'-keto form, the closed active site is "sealed off" from the environment, as indicated by a large (10(8)(-)(9)-fold) reduction in the rate of departure of ligands, a feature that prevents exposure of the labile 3'-keto intermediates to the aqueous environment. Elimination of the 5'-substituent (Hcy in the hydrolytic direction, water in the synthetic direction) generates the central intermediate 4',5'-didehydro-5'-deoxy-3'-ketoadenosine. Abortive 3'-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redox catalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is 10(4)-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constant similar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redox catalytic power appears to be a conformationally induced increase in the distance across which hydride transfer must occur between cofactor and substrate, the responsible conformational change again being that which "seals" the active site. The crystal structure reveals a well-defined chain of three water molecules leading from the active site to the subunit surface, which may serve as a relay for proton exchange between solvent and active site in the closed form of the enzyme, permitting maintenance of active-site functional groups in catalytically suitable protonation states.

Crystal structures of Mycobacterium tuberculosis S‐adenosyl‐L‐homocysteine hydrolase in ternary complex with substrate and inhibitors

“…by molecular replacement method, EPMR, using the human SAHH model (PDB ID: 1LI4) as a search model for the extending data from 25 to 3.5 Å (Yang et al 2003). Complexes with DZA, 2FA, and ARI were solved by molecular replacement from the ADO complex.…”

“…The crystal structure for SAHH has been solved for three different organisms to date, all eukaryotic: human, rat (Rattus rattus), and malaria (Plasmodium falciparum) (Turner et al 1998;Hu et al 1999;Komoto et al 2000;Huang et al 2002;Takata et al 2002;Yang et al 2003;Tanaka et al 2004;Yamada et al 2005Yamada et al , 2007. The proposed SAHH catalytic mechanism involves a sequence of steps as follows (Fig.…”

“…For example, 29,39-dihydroxycyclopent-49-enyl adenine (DHCeA) inhibits SAHH via a type-I mechanism (Wolfe and Borchardt 1991) by oxidation of the 39 hydroxyl to a 39 keto group, which reduces the bound NAD + to NADH, thus inactivating the enzyme (Turner et al 1998). This compound represents a family of carbocyclic pyrimidine nucleosides, like neplanocin A (Mosley et al 2006), that form similar chemically abortive complexes (Yang et al 2003). ARI is also expected to inhibit SAHH via a type-I mechanism (Liu et al 1996).…”

A Tribute to Ronald T. Borchardt—Teacher, Mentor, Scientist, Colleague, Leader, Friend, and Family Man

“…[113][114][115][116][117][118][119][120][121][122][123] The mechanism by which designed adenosine analogs inactivate AdoHcy hydrolase was also elucidated and provided better understanding of the enzyme's active site and catalytic mechanism. 112,119,[124][125][126][127][128][129][130][131][132][133] These advances were made possible through long standing and productive collaborations with Ron's fellow KU faculty members Professors Richard Schowen and Krzysztof Kuczera, and Professor P. Lynne Howell at the University of Toronto.…”

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

“…Data were collected at beamline 7.2 at the Stanford Synchrotron Radiation Light Source and processed with HKL2000 (37). Molecular replacement was performed with PHASER (38) using the structure of human SAHH as a search model (39) and diffraction data extending to 2.8 Å. Two independent subunits of SAHH were found in the crystallographic asymmetric unit with a final translational z-score of 66.4.…”