A complex of methylthioadenosine/S-adenosylhomocysteine nucleosidase, transition state analogue, and nucleophilic water identified by mass spectrometry

Abstract

An enzyme-stabilized nucleophilic water molecule has been implicated at the transition state of Escherichia coli methylthioadenosine nucleosidase (EcMTAN) by transition state analysis and crystallography. We analyzed the EcMTAN mass in complex with a femtomolar transition state analogue to determine whether the inhibitor and nucleophilic water could be detected in the gas phase. EcMTAN-inhibitor and EcMTAN-inhibitor-nucleophilic water complexes were identified by high-resolution mass spectrometry under nondenaturing conditions. The enzyme-inhibitor-water complex is sufficiently stable to exist in the gas phase.

Figure 1

The transition state structure of MTA catalyzed by EcMTAN.

Figure 2

The complex of VcMTAN, BuT-DADMe-ImmA and a water molecule. A. The chemical structure of BuT-DADMe-ImmA is shown in the orientation corresponding to its position in the crystal structure. B. In the crystal structure (pdb 3DP9), BuT-DADMe-ImmA (green) and a water molecule (red dot) are surrounded by active site residues (gray). Inset: hydrogen bonding network to the water molecule. PYMOL was used to generate the graphs.

Figure 3

MS analysis of EcMTAN in complex with stoichiometric BuTDADMe-ImmA. Inset: with increased focusing potential and declustering potential, water molecules are shifting out of the enzyme-inhibitor complex.