Methionine aminopeptidase (MetAP) is a ubiquitous metallohydrolase responsible for the removal of N-terminal methionine in nascent peptides. MetAP's active site contains a high-affinity and a low-affinity metal binding site, and both mono- and dimetallic MetAP mechanisms have been proposed in the literature. This study aims to resolve the debate on enzyme mechanism by exploring the effect of inhibitor binding on the  of the low-affinity site. We have developed a method to quantitatively calculate the dissociation constants of cobalt in MetAP in the presence of inhibitors by using equilibrium dialysis. Preliminary results show that in the absence of inhibitor, Kd1 = 6 � 3 �M and Kd2 = 22 � 18 �M. In the presence of inhibitor PDA3, Kd1 = 5 �  1 �M and Kd2 = 13 �6 �M. Such findings implicate a dimetallic mechanism with no cooperative metal binding. Further study is needed to validate results.

Active site metal binding in inhibitor complexes of methionine aminopeptidase

243rd National Meeting (2012)

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