In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.

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The protein proopiomelanocortin (POMC) is a precursor several nerve peptide hormones, including MSHs, ACTH, CLIP, LPH, b-endorphin, and the Joining peptide (JP). The biological activity of POMC derived peptides have been well studied except for JP. To investigate the biological function of JP, we chemically synthesized JP by an Fmoc solid phase method. However, the yield of the synthesized JP was poor and a highly efficient a/b-rearrangement in the Asp-Gly sequence was observed. Therefore, we evaluated the rearrangement of the Asp-Gly sequence under several conditions to estimate the biological activity and the stability of the correct configuration of JP. For this purpose, JP was chemically synthesized by ordinary Fmoc and Boc solid phase methods. After deprotection, JP was separated and identified by reverse-phase HPLC and MALDI-TOF/MS, respectively. The rearrangement of the Asp-Gly moiety was observed in the case of the Fmoc method but was not significant in the case of the Boc method. To estimate the stability of the Asp-Gly moiety, JP was treated with several buffers in the pH range of 1-8. The a/b-rearrangement was gradually increased in a pH-dependent manner and was significantly observed under strongly acidic conditions. In addition, salt effects for the rearrangements were also estimated. The results will be discussed in this paper.