Abstract
We have performed a computational study of different protomeric states of the methionine aminopeptidase active site using a combined quantum-mechanical/molecular mechanical simulation approach. The aim of this study was to clarify the native protonation state of the enzyme, which is needed for the development of novel irreversible inhibitors that can possibly be used as antiangiogenic and antibiotic drugs by virtual screening and other drug design methods. The results of the simulations indicated that two protonation states are possible without disturbing the overall geometry of the active site. We then verified experimentally the presence of the two protonation states by studying the substrate hydrolysis and inhibitor binding reactions at different pH values and come to the conclusion that one of the protomeric states is relevant for inhibitor binding, whereas the other is relevant for substrate hydrolysis. This result has implications for the development of other inhibitors of this class of enzymes and adds a new perspective to the pharmacological properties of the antiangiogenic drug fumagillin, which is an irreversible inhibitor of the human methionine aminopeptidase type II.
Highlights
Methionine aminopeptidases (MetAPs)1 play a central role for in vivo protein synthesis as they remove the starter methionine from newly synthetized proteins
Simulations—In the CPMD simulations, the system with the two water molecules coordinated to the zinc ions showed a pronounced movement of the coordinating water molecules away from the x-ray structure of E. coli MetAP that has one water molecule located between the metals
An active site with two water molecules coordinated to the metal ions is stable, albeit with a different coordination geometry
Summary
Methionine aminopeptidases (MetAPs) play a central role for in vivo protein synthesis as they remove the starter methionine from newly synthetized proteins. The antiangiogenic effect of fumagillin and other inhibitors of MetAP-II have been attributed to the inhibition of the Ets-1 transcription factor expression and the activation of the. Several three-dimensional structures of MetAPs have been determined by x-ray diffraction methods including the structure of human MetAP-II with a covalently bound fumagillin molecule [13,14,15]. Our motivation for examining the MetAPs from a theoretical point of view stems from the highly selective, irreversible inhibition mechanism of fumagillin and related epoxides. We believe that the fumagillin/MetAP example is a good test case for theoretical methods that aim at rationalizing the development of covalent enzyme inhibitors, because a large amount of high quality x-ray structural data, with bound inhibitors, has been published over the last few years
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