RESUMEN
Aminoacyl-tRNA synthetases catalyze the stepwise coupling of specific amino acid substrates to their cognate tRNAs. The first intermediate formed in this process is the aminoacyl-adenylate, which then subsequently reacts with the 3'-terminus of the cognate tRNA to transfer the amino acid to the tRNA. This overall reaction is critical for protein biosynthesis and is quintessential to the viability of all organisms. Therefore, the selective inhibition of bacterial amino acid-tRNA synthetases is the focus of intense current interest for the development of novel antibacterial agents. In order to elucidate some of the critical factors involved in recognition and binding of potential inhibitors to these bacterial systems, the current report has focused on the methionyl-tRNA synthetase from Escherichia coli. This enzyme has been studied with two sets of bioisosteric replacements in the methionine and methionyl-adenylate structures. Replacements of the carboxyl group of methionine with the phosphinic and phosphonic acid moieties were used to probe the effects of including potential transition state analogs on enzyme inhibition. The contributions of the aminoacyl-adenylate structure and the effect that fluorination has on inhibitory activity were investigated utilizing 5'-O-[(L-methionyl)-sulfamoyl]adenosine and 5'-O-[(S-trifluoromethyl-L-homocysteinyl)-sulfamoyl]adenosine. The K(i) values for these compounds were determined to be 0.4 mM, 1.2 mM, 0.25 nM and 2.4 nM respectively. A discussion of this data in relation to structural information provided by the recent determination of the three-dimensional structures of the E. coli enzyme with several of these compounds is presented.
Asunto(s)
Adenosina/análogos & derivados , Antibacterianos/química , Inhibidores Enzimáticos/química , Homocisteína/análogos & derivados , Metionina-ARNt Ligasa/antagonistas & inhibidores , Metionina/análogos & derivados , Adenosina/química , Adenosina/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/farmacología , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Homocisteína/química , Homocisteína/farmacología , Metionina/química , Metionina/farmacología , Metionina-ARNt Ligasa/química , Metionina-ARNt Ligasa/genética , Organofosfonatos/química , Ácidos Fosfínicos/química , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography. Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79. In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79. The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor. The mode of interaction of His79 is, however, different. In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues. In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues). These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79. The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase.
Asunto(s)
Aminopeptidasas/metabolismo , Escherichia coli/enzimología , Sustitución de Aminoácidos , Cristalografía por Rayos X , Enlace de Hidrógeno , Metionina/análogos & derivados , Metionina/metabolismo , Metionil Aminopeptidasas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Fósforo , Conformación Proteica , Programas Informáticos , Relación Estructura-ActividadRESUMEN
Hydroxamate-containing tripeptide analogs resembling a reactive intermediate in glyoxalase I catalysis were prepared by solution methods and were found to be competitive inhibitors of the enzyme from Saccharomyces cerevisiae. Electronic properties of the hydroxamate functionality as well as those of the expected intermediates in the enzyme-catalyzed reaction were compared.