The bioorganic chemistry of transglutaminase — from mechanism to inhibition and engineering

Abstract

Through a multidisciplinary approach comprising organic synthesis, molecular biology, and physical organic kinetic studies, we have studied the mechanism of transglutaminase-mediated transamidation. More recently, we have applied our understanding of the mechanism to the design of reversible inhibitors and affinity labels for biological application. We have also undertaken the engineering of transglutaminase for its application as a “peptide synthase”. Herein, we present a brief overview of previously published work as well as recent results presented at the 2007 Merck–Frosst Centre for Therapeutic Research Award Lecture.Key words: transglutaminase, enzyme mechanism, enzyme inhibition, protein engineering

Transglutaminase Is Required for Epidermal Squamous Cell Carcinoma Stem Cell Survival

“…Spheroids were grown for 8 d and then treated with NC9, an irreversible inhibitor of TG2 transamidase (TGase, crosslinking) activity (16). NC9 treatment reduced spheroid number (Fig.…”

“…6G). NC9 is an irreversible inhibitor that covalently binds to TG2 to inactivate TGase activity (16). However, NC9 also locks TG2 into an extended conformation (38) which is associated with inactivation of GTP binding (63), as TG2 GTP binding requires a closed configuration (63).…”

“…Peroxidase-conjugated anti-mouse IgG (NXA931) and anti-rabbit IgG (NA934V) were obtained from GE Healthcare (Buckinghamshire, UK). Production of NC9 was described previously (16). TG2- (sc-37514) and control-siRNA (sc-37007) were purchased from Santa Cruz (Dallas, TX).…”

N,N′‐Methylenediimidazolium Salts: From Self‐Assembly to an Efficient DNAse Protection System

“…MilliQ water was used for the electronic microscopy assays and the DNA studies. DNase I was obtained from Invitrogen and plasmid pQE32-gTG [14] from Joelle N. Pelletier (UniversitØ de MontrØal). 1 H and…”

“…To test this hypothesis, complexation assays with linear DNA were performed. [14] DNA complexation was undertaken with a linear DNA fragment obtained by digestion of the circular pQE32-gTG plasmid with the restriction enzyme Xho1. The linearized pQE32-gTG DNA was incubated for 30 min at 37 8C with 50 mm of 2 or 3 or with 100 mm of 4.…”