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The mode of inhibition of mechanism-based inhibitor (2,Ki = 0.4 (mu)M) of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which is designed to mimic the combined key features of its natural substrates arabinose-5-phosphate and phoshoenolpyruvate into a single molecule, is investigated. A novel frequency-selective rotational-echo double-resonance (REDOR) experiment is developed and applied.

Results demonstrate that the enzyme binds phosphoenolpyruvate and arabinose-5-phosphate via arginine and lysine residues by different mechanisms. Data reveal that the active site residues for phosphoenolpyruvate are structurally rigid residues lysine-138 and arginine-168 wheras arginine 63, which binds to arabinose-5-phosphate is flexible.

Chemical research shows that the spin transition of oxidized metalloporphyrin manganese porphyrin cation radical (TMP.+)MnIII(ClO4)2 is modified at high temperatures from S = 3/2 to S = 5/2. Other iron porphyrins exhibit similar characteristics. The production of (TMP)Mn(super 1V)(Cl)2 involves the ligand exchange of perchlorato ligands in (TMP.+)Mn1V(Cl)2.

Research is presented describing the identification of active site residues of the enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase by the use of rotational-echo double-resonance solid-state NMR experiments.

In this report the mode of inhibition of mechanism-based inhibitor (2, K[sub i] = 0.4 µM) of 3-deoxyD-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which was designed to mimic the combined key features of its natural substrates arabinose-5-phosphate (A5P) and phoshoenolpyruvate (PEP) into a single molecule, was investigated. Our earlier solid-state NMR observations identified the inhibitor to bind in a way that partly mimics ASP, while the phosphonate moiety of its PEP-mimicking part exhibits no interactions with enzyme residues. This result was apparently in disagreement with the competitive inhibition of 2 against PEP and with the later solved crystal structure of KDO8PS-2 binary complex identifying the interactions of its PEP-mimicking part with the enzyme residues that were not detected by solid-state NMR. To solve this discrepancy, further solid-state REDOR NMR and [sup 31]P solution NMR experiments were applied to a variety of enzyme complexes with the substrates and inhibitor. In particular, a novel frequency-selective REDOR experiment was developed and applied. Integration of the solution and solid-state NMR data clearly demonstrates that under conditions of stoichiometric enzyme-ligand ratio at thermodynamic equilibrium (a) PEP binding is unperturbed by the presence of 2 and (b) both PEP and 2 can bind simultaneously to the synthase, i.e., form a ternary complex with PEP occupying its own subsite and 2 occupying A5P's subsite. The latter observation suggests that under the conditions used in our NMR measurements, the inhibition pattern of 2 against PEP should have a mixed type character. Furthermore, the NMR data directly demonstrate the distinction between the relative binding strength of the two moieties of 2: enzyme interactions with PEP-mimicking moiety are much weaker than those with the A5P moiety. This observation is in agreement with KDO8PS-2 crystal structure showing only remote contacts of the phosphonate due to large structural... [ABSTRACT FROM AUTHOR]