Transition State Analysis of the Aldol-Like Reaction Catalyzed by NeuB, a Sialic Acid Synthase, Reveals a Stepwise Cationic Mechanism

The sialic acid synthase NeuB, and other α-carboxyketose synthases, are long-standing targets for inhibition by potential antimicrobial compounds. NeuB catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) that passes through a tetrahedral intermediate (THI) to form N-acetylneuraminic acid. We measured multiple kinetic isotope effects (KIEs) in order to determine the transition state (TS) structure of the NeuB-catalyzed reaction. As part of this study, the use of incomplete T ₁ relaxation during KIE measurement by NMR was investigated, and then used to accelerate KIE measurements 9-fold. KIEs measured at the 3-¹³C, 2-¹³C and 2-¹⁸O positions in PEP reveal a stepwise mechanism of THI formation in which the PEP C3 to ManNAc C1'(aldehyde) bond (C3···C1') is formed first, leading to a cationic intermediate. However, the transition state is so early in C3···C1' bond formation, with a C3···C1' bond order of 0.17 to 0.35, that there is little cationic character in the transition state. The lack of change in geometry or charge in forming the transition state implies that NeuB may function primarily by "catalysis-by-approximation"; i.e., its main contribution to TS stabilization is bringing the reactants together in the correct orientation. It also implies that inhibitors designed to mimic a positive charge are unlikely to be effective. Instead, neutral inhibitors that maximize the number of active site interactions are more likely to be effective.