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Substrate analogues as mechanistic probes for 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase and 3-Deoxy-D-manno-octulosonate 8-phosphate synthase : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7P synthase) catalyses the condensation reaction between phosphoenolpyrtivate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (D-E4P). 3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase) catalyses a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (D-A5P). These enzymes are two functionally unrelated enzymes that share many mechanistic and structural features. D-Threose 4-phosphate (D-T4P), L-threose 4-phosphate (L-T4P), D-arabinose 5-phosphate (D-A5P), D-lyxose 5-phosphate (D-L5P), and L-xylose 5-phosphate (L-X5P) have been prepared synthetically or enzymatically to provide insights into aspects of metal requirement and substrate specificity. These compounds were different stereoisomers of natural substrates D-E4P and D-A5P. The results presented in this thesis show that D-T4P and L-T4P (C2 and C3 stereoisomers of D-E4P) are substrates for the DAH7P synthases from E. coli and P. furiosus. For N. meningitidis KDO8P synthase, natural substrate D-A5P and L-X5P (the C4 epimer of D-A5P) were substrates, whereas D-L5P, the C3 epimer of D-A5P, was not. These observations show that the configuration of the C2 and C3 hydroxyl groups is not important for DAH7P synthase reaction, but having the correct configuration at these positions is critical for KDO8P synthase. The analysis of the interaction of D-T4P and L-T4P with DAH7P synthase, and D-A5P, D-R5P, and L-X5P reveals previously unrecognised mechanistic differences between the DAH7P synthase-catalysed reaction and that catalysed by the closely related enzyme, KDO8P synthase.