Date of Award
Master of Science (MSc)
Paul J. Berti
Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid N-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to N-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn2+. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH2OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (Ki*) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH2OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuBIS), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuBRS), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-14C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuBRS is required for complete inhibition.
Popović, Vladimir, "Inhibition of the bacterial sialic acid synthase, NeuB" (2012). Open Access Dissertations and Theses. Paper 6820.
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