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Published online before print March 9, 2004, 10.1110/ps.03433304
Protein Science (2004), 13:979-991. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Roberto A. Chica, Paul Gagnon, Jeffrey W. Keillor and Joelle N. Pelletier

Département de chimie, Université de Montréal, Montréal, Québec, Canada H3C 3J7

(RECEIVED September 11, 2003; FINAL REVISION November 27, 2003; ACCEPTED December 1, 2003)



Abstract

Transglutaminases (TGases) catalyze the cross-linking of peptides and proteins by the formation of {gamma}-glutamyl-{varepsilon}-lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide-based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using a combined kinetic/molecular modeling approach, we have generated a model for the binding of small acyl-donor peptide substrates to tissue TGase from red sea bream. Kinetic analysis of various N-terminally derivatized Gln-Xaa peptides has demonstrated that many CBz-Gln-Xaa peptides are typical in vitro substrates with KM values between 1.9 mM and 9.4 mM, whereas Boc-Gln-Gly is not a substrate, demonstrating the importance of the CBz group for recognition. Our binding model of CBz-Gln-Gly on tissue TGase has allowed us to propose the following steps in the acylation of tissue TGase. First, the active site is opened by displacement of conserved W329. Second, the substrate Gln side chain enters the active site and is stabilized by hydrophobic interaction with conserved residue W236. Third, a hydrogen bond network is formed between the substrate Gln side chain and conserved residues Y515 and the acid-base catalyst H332 that helps to orient and activate the {gamma}-carboxamide group for nucleophilic attack by the catalytic sulphur atom. Finally, an H-bond with Y515 stabilizes the oxyanion formed during the reaction.

Keywords: tissue transglutaminase; molecular modeling; docking; enzyme kinetics; peptide substrate; autodock; TGase

Reprint requests to: Joelle N. Pelletier, Département de chimie, Université de Montréal, 2900 Édouard-Montpetit, Montréal, Québec, Canada H3C 3J7; e-mail: joelle.pelletier{at}umontreal.ca; fax: (514) 343-7586.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03433304.


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