Substrate Binding Stabilizes S-Adenosylhomocysteine Hydrolase in a Closed Conformation

Abstract

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.

Binding of Cu2+ to S-adenosyl-l-homocysteine hydrolase

“…Therefore, the rate of Cu 2þ getting into its sites will be determined by the frequency of openingclosing of the enzyme structure. The frequency of domain movement has been determined to be 25 ns [27], which suggests a k on value of less than 4 Â 10 7 M À1 s À1 . This is in good agreement with the experimental result of 7 Â 10 6 M À1 s À1 .…”

The Rationale for Targeting the NAD/NADH Cofactor Binding Site of Parasitic S-Adenosyl-L-Homocysteine Hydrolase for the Design of Anti-Parasitic Drugs

“…The open conformation of SAHH enables substrate binding and the closed conformation provides a closer contact between substrate and cofactor in the active site, as is needed for catalysis. The enzyme converts back to the open conformation upon product release 21 .…”

“…The catalytic cycle involves two enzyme conformational states (Protein Data Bank code: 1KY4 20 and 1A7A 1, 21 ): an open-closed interconversion of each monomer, which regulates substrate binding and product release, and a ~14° rotation of one dimer relative to the second dimer, which leads to a reduction in the volume of the tetramer and may serve to “seal” the closed form. The “sealed” active sites help to prevent contact with the environment of intermediates among a series of transition states 21, 25 .…”

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.…”