UBC Theses and Dissertations
An investigation of the mechanism of sialic acid synthase Simard, Dave
Sialic acid synthase (SAS) catalyzes the aldol-like condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) resulting in the formation of Nacetylneuraminic acid (NeuNAc). The neuB gene, coding for SAS, was cloned out of the Neisseria meningitidis genome and inserted into the pET-30 Xa/LIC vector, thus adding a His-tag onto the gene product. The His-tagged protein (SASHIS) was purified and kinetically characterized using a novel continuous coupled UV assay. SAS is a slow enzyme as can be seen by its k[sub cat] of 0.8 ± 0.1 s⁻¹. Furthermore, it binds to PEP more tightly than it does to ManNAc, K[sub M] 0.25 ± 0.07 mM and 9.4 ± 1.4 mM, respectively. These kinetic values for SASHIS are in reasonable agreement with literature values obtained with the wild type enzyme. The mechanism employed by SAS to catalyze the condensation reaction was unknown. Through NMR and ¹⁸O-labelled substrate experiments, it was shown that the synthase releases the phosphate of PEP through C-O bond cleavage during the condensation with ManNAc. Thus SAS uses a mechanism analogous to DAHP and KDO-8P synthases, other enzymes forming higher order sugars.
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