Mechanism of action of guinea pig liver transglutaminase. VII. Chemical and stereochemical aspects of substrate binding and catalysis.

Active ester Cp-nitrophenyl ester) hydrolysis and transfer reactions catalyzed by transglutaminase have been studied at pH 7.5 and 0” in the presence of 50 mu Ca++. The initial velocities of appearance of two products of the reactions, $-nitrophenol and N-acylamines, were found to be consistent with a ping pong mechanism in which acyl-enzyme formed from active ester may react with water (hydrolysis) or with primary amine (transfer). This 6nding, in conjunction with previous evidence that the hydrolysis and transfer of transglutaminase-catalyzed glutamine substrate conform to this mechanism, gives strong support for an acyl-enzyme theory of transglutaminase action. The D and L antipodes of several active ester and amide (6rst) substrates and the D and L forms of the amine (second) substrate, alanine ethyl ester, have been employed as a means of detecting stereochemical dissymmetry within the active site of transglutaminase. Notable difFerences in the kinetic parameters of the reactions of the D and L antipodes of benzyloxyca.rbonyl(Z)-alanine p-nitrophenyl ester and those of Z-a-glutamyl(y-p-nitrophenyl ester)glycine point up dissymmetric arrangements of groupings within the active center of the activated native enzyme. The differences in reactivities of Dand L-alanine ethyl esters in the transglutaminase-catalyzed transfer reaction of various first substrates, both ester and amide, indicate variation in the spatial arrangement of groupings within the amine site of the different acyl-enzymes. This is manifest as an acylenzyme-dependent preference for one antipode over the other. The almost absolute stereospecificity of transglutaminase toward glutamine substrate is evident in its strong preference for the L form of Z-glutaminylglycine. However, the enzyme displays substantial activity toward both D and L isomers of Z-cr-aminoadipamylglycine (Z-homoglutaminylglycine). These findings provide a preliminary approach to an understanding of the spatial relationships between the substrate and the enzyme.