It has been frequently observed that the inhibitors developed against metalloenzymes harboring non-physiological metal ions (via in vitro enzymatic screening), are ineffective under in vivo conditions. The identity of physiological metal ions for pathogenic metalloenzymes is important not only from the point of deducing their mechanistic details but also for designing their potent inhibitors as therapeutic agents. In other words, the kinetically derived activation constants (K a’s) of metal ions are taken to be the measure of their direct binding affinities (K d’s), and such assumption may not be true for all metalloenzymes and their cognate metal ions. Unfortunately, the assignment of physiological metal ions to the latter metalloenzymes relies on the assumption that the enzyme substrates (under steady-state condition) do not alter the intrinsic binding affinities of those metal ions. Based on their binding affinities, metalloenzymes typically fall in two categories : (i) The enzymes which interact with their physiological metal ions fairly tightly (K d 10 nM), and such enzymes usually lose their metal ions during the course of purification. The drug discovery endeavour involving pathogenic metalloenzymes has often been hampered (at least in part) due to difficulty in identifying their physiological metal ions. On accounting for the binding affinity vis a vis the catalytic efficiency of the enzyme for different metal ions, it appears evident that that the “coordination states” and the relative softness” of metal ions are the major determinants in facilitating the EcMetAP catalyzed reaction. However, the enzyme-metal binding data did not adhere to the Irving-William series. The experimental data revealed that among all metal ions, Fe 2+ exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme. Due to competitive displacement of the enzyme-bound Eu 3+ by different metal ions, we could determine the binding affinities of both “activating” and “non-activating” metal ions for the enzyme via fluorescence spectroscopy. coli methionine aminopeptidase ( EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.We report herein, for the first time, that Europium ion (Eu 3+) binds to the “apo” form of E.
METAL ION BONDING METHIONINE FULL
In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents.
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