Aromatic-aromatic interactions play an important role in the enzyme-substrate recognition mechanism and in stabilization of proteins. Gelonin - a ribosome inactivating protein (RIP) from the plant Gelonium multiflorum - belongs to type-I RIPs and shows N-glycosylation activity which has been used as a model to explain the role of aromatic-aromatic stack pairing in RIPs. RIPs have a different substrate binding site and catalytic site. Role of tyrosine residues at the binding site has already been known but the role of tyrosine residues at catalytic site is still unclear. In this study, the role of tyrosine-adenine-tyrosine aromatic stack pairing at the catalytic site was studied by in silico mutation studies using molecular dynamic simulations. Through this study we report that, despite the fact that aromatic stack pairing aids in recognition of adenine at binding site, both the tyrosine residues of stack pairing play a crucial role in the stabilization of adenine at catalytic site. In the absence of both the tyrosine residues, adenine was unstable at catalytic site that results in the inhibition of N-glycosylation activity of gelonin protein. Hence, this study highlights the importance of pi-pi stack pairing in the N-glycosidic activity of gelonin by determining its role in stabilizing adenine at catalytic site. (C) 2011 Elsevier Inc. All rights reserved.