Factor XIII (FXIII) catalyzes formation of γ-glutamyl-ε-lysyl crosslinks between reactive glutamines (Q) and lysines (K). In plasma, FXIII is activated proteolytically (FXIII-A*) by the concerted action of thrombin and Ca2+. Cellular FXIII is activated non-proteolytically (FXIII-A°) by elevation of physiological Ca2+ concentrations. FXIII-A targets plasmatic and cellular substrates, but questions remain on correlating FXIII activation, resultant conformational changes, and crosslinking function to different physiological substrates. To address these issues, the characteristics of FXIII-A* versus FXIII-A° that contribute to transglutaminase activity and substrate specificities were investigated. Crosslinking of lysine mimics into a series of Q-containing substrates were measured using in-gel fluorescence, mass spectrometry, and UV-Vis spectroscopy. Covalent incorporation of fluorescent monodansylcadaverine revealed that FXIII-A* exhibits greater activity than FXIII-A° toward Q residues within Fbg αC (233-425 WT, Q328P Seoul II, and Q328PQ366N) and actin. FXIII-A* and FXIII-A° displayed similar activities toward α2-antiplasmin, fibronectin, and Fbg αC (233-388, missing FXIII binding site αC 389-402). Furthermore, the N-terminal α2-AP peptide (1-15) exhibited similar kinetic properties for FXIII-A* and FXIII-A°. MALDI-TOF mass spectrometry assays with glycine ethyl ester and Fbg αC (233-425 WT, αC E396A, and truncated αC (233-388) further documented that FXIII-A* exerts greater benefit from the αC 389-402 binding site than FXIII-A°. Conformational properties of FXIII-A* versus A° are proposed to help promote transglutaminase function toward different substrates. A combination of protein substrate disorder and secondary FXIII binding site exposure are utilized to control activity and specificity. From these studies, greater understandings of how FXIII-A targets different substrates are being achieved.
model enzyme typical for this class of cellobiohydrolases (CBH). It processively releases many sugar molecules without dissociating from the substrate. However, dissociation from the cellulose substrate to avoid barriers along the fibril has been shown to be rate-limiting with an unknown mechanism. Using equilbrlium classical equilibrium molecular dynamics simulations and Hamiltonian replica exchange techniques, we explore two potential dissociation mechanisms. Kinetics estimates determined through computation suggest that stepwise dethreading of the cellulose fibril from the enzyme binding tunnel is four orders of magnitude faster than an alternative mechanism whereby the loops surrounding the binding tunnel open and the bound substrate dissociates through the gap. When coupled to dissociation rate experiments enabled by a tunnel-locking mutant indicating equivalent dissociation rates, the stepwise dethreading mechanism likely predominates for enzyme dissociation in TrCel7A and similar processive CBH enzymes.
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