One- and Two-Proton Transfer Mechanisms Coexist in One Active Site

Area of Science:

• Biochemistry
• Plant Science
• Computational Chemistry

Background:

• Acibenzolar-S-methyl (ASM) is a key commercial plant activator for systemic acquired resistance (SAR).
• The precise catalytic mechanism of ASM hydrolysis by salicylic acid binding protein 2 (SABP2) has remained unclear.
• Understanding enzyme-substrate interactions is crucial for plant defense mechanisms.

Purpose of the Study:

• To elucidate the fundamental catalytic mechanism of SABP2-mediated hydrolysis of ASM.
• To investigate the differences in SABP2's catalytic mechanisms with varying substrates.
• To provide insights for the development of next-generation plant SAR activators.

Main Methods:

• Quantum Mechanics/Molecular Mechanics (QM/MM) simulations.
• Charge transfer analysis.
• Small-molecule synthesis and biochemical assays (HPLC).

Main Results:

• SABP2 employs a two-proton transfer mechanism for ASM hydrolysis, with charge transfer stabilizing the intermediate.
• For ASM analogue BTM, SABP2 uses a one-proton transfer mechanism, stabilizing the tetrahedral intermediate via electrostatic effects.
• Computational findings were validated by comparable HPLC analyses of SABP2 esterase activity.

Conclusions:

• SABP2 exhibits catalytic promiscuity, utilizing distinct mechanisms for different substrates.
• The study clarifies enzyme catalytic mechanisms and provides a foundation for designing novel plant SAR activators.
• This research advances the understanding of enzyme function and plant immune responses.