MetAP is a member of the metalloaminopeptidase family. coli MetAP that inhibit different metalloforms of the enzyme with remarkable selectivity ( 18) they may become powerful tools to define the intrinsic metal used by MetAP under physiological conditions. Our laboratory has recently discovered several classes of in vitro inhibitor for E. The former has most often been Co(II), whereas Mn(II) and Fe(II) have been suggested for the latter ( 15, 17). Thus, another reason for the apparent lack of antibacterial and antiangiogenic activities of some potent in vitro MetAP inhibitors may be a mismatch between the metal ion used for activity measurement in vitro and the metal ion used to activate the apoenzyme inside cells. It is not known which of these ions may be present in the MetAP under physiological conditions. It has long been known that MetAPs can be activated in vitro by a number of different divalent metal ions, including Mn(II), Fe(II), Co(II), Ni(II), and Zn(II) ( 14– 16), and we have shown that inhibitors of the Co(II) form may or may not inhibit MetAP in other metalloforms ( 14). One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to reach their intended target, or perhaps they are efficiently transported back out of the cells. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial ( 10– 12) or antiangiogenic ( 13) activities. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Bengamides inhibit both types of MetAP ( 9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 ( 6– 8). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes ( 5). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents ( 4). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli ( 2) and Salmonella typhimurium ( 3). In a significant number of cases, this initiating methionine residue is removed, either co- or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) ( 1). 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.Īll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG.
#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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