Mechanism of action of guinea pig liver transglutaminase. IV. The trimethylacyl enzyme.

The transglutaminase-catalyzed hydrolysis and transfer reactions have been studied kinetically in the forward direction at pH 8 in the presence of a 0.1 M level of the activating metal ion, Ca++. Isotope exchange and product inhibition studies were also conducted. The initial velocity, product inhibition, and isotope exchange patterns are consistent with a ping pong mechanism in which (a) glutamine substrate binds to enzyme in a binary complex, (b) ammonia dissociates with the formation of an acyl enzyme intermediate, and (c) acyl group is transferred from enzyme either to acceptor amine to form y-glutamyl-amine product (transfer), or to water to form glutamic acid product (hydrolysis). A gel filtration study that shows binding of amine to enzyme only in the presence of glutamine substrate complements this proposed ping pong mechanism. Values have been estimated for all except two of the Michaelis and dissociation constants involved in combination of substrates and products with the enzyme forms, i.e. free or acyl enzymes, with which they bind. The transfer reaction in the reverse direction has been shown chromatographically.