|M.Sc Student||Rabkin Emilia|
|Subject||Design, Synthesis and Evaluation of 1-Deoxy Analog of|
Arabonose-5-Phosphate as Inhibitor of KDO8P
|Department||Department of Chemistry||Supervisor||Professor Timor Baasov|
Catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, an enzyme involved in the biosynthesis of lipopolysaccarides of Gram-negative bacteria, remains a fascinating subject for both bioorganic and medicinal researches. The enzyme catalyses an adol-type condensation of D-arabinose 5-phosphate (A5P) with phosphoenol pyruvate (PEP) to produce the eight-carbon saccharide KDO8P and inorganic phosphate (Pi).
The structure and mechanism of KDO8P synthase have actively studied during last decade as this enzyme represents an important target for antibiotic therapy. Previous studies have demonstrated that the enzyme acts upon acyclic form of A5P. Very recently the binding properties of both substrates to the enzyme were investigated by solid-state REDOR NMR. These studies have demonstrated that the enzyme recognizes and binds both cyclic anomers (α and β) of A5P. In attempts to understand the binding properties of A5P, and in parallel to design new inhibitor of the enzyme, cyclic 1-deoxy analog of A5P, 1dA5P, that is chemically prohibited to ring opening, was synthesized and evaluated as inhibitor of the enzyme.
Kinetic evaluation of 1dA5P defined the analog as competitive inhibitor against A5P binding with inhibition constant, Ki, of 0.6 mM. The ability of 1dA5P to compete with A5P supports the hypothesis that the furanose form of A5P can bind to the enzyme. No inhibition was found against PEP binding.
Employing 31P and 13C solution NMR we have shown that 1dA5P forms the binary E•1dA5P and the ternary E•1dA5P•PEP complexes. In the latter, the binding of PEP is pronouncedly different than its binding in the binary complex E•PEP.
Combination of the kinetic and NMR data leads us to the conclusion that the kinetic mechanism is rapid equilibrium random mechanism, according to which the order of substrate binding is determined by their binding constants.