Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism

Guinea pig liver transglutaminase (TGase) reacts with 0.1 mM N-Cbz-L-Glu(ga mma -p-nitrophenyl ester)Gly (5, prepared herein, K-M = 0.02 mM) to undergo rapid acylation that can be followed spectrophotometrically at 400 nm (pH 7.0, 25 degreesC). Deacylation of the transiently formed thiolester acyl en zyme intermediate via catalytic aminolysis was studied in the presence of s ix primary amines of widely varying basicity (PKNH+ = 5.6-10,5), Steady-sta te kinetic studies were performed to measure k(cat) and K-M values for each amine substrate. A Bronsted plot constructed through the correlation of lo g(k(cat)/K-M) and PKNH+ for each amine substrate displays a linear free-ene rgy relationship with a slope beta (nuc) = -0.37 +/- 0.08. The shallow nega tive slope is consistent with a general-base-catalyzed deacylation mechanis m in which a proton is removed from the amine substrate during its rate-lim iting nucleophilic attack on the thiolester carbonyl, Kinetic isotope effec ts were measured for four acceptor substrates (water, kie 1.1 +/- 0.1; amin oacetonitrile, kie = 5.9 +/- 1.2; glycine methyl ester, kie = 3.4 +/- 0.7; N-Ac-L-lysine methyl ester, kie 1.1 +/- 0.1) and are consistent with a prot on in flight at the rate-limiting transition state. The active site general -base implicated by these kinetic results is believed to be His-334, of the highly conserved TGase Cys-His-Asp catalytic triad.