Mechanism of Action of Guinea Pig Liver Transglutaminase

Abstract

Abstract Evidence is presented that purified guinea pig liver transglutaminase catalyzes the hydrolysis of p-nitrophenyl trimethylacetate. As in the case of the other transglutaminase-catalyzed reactions, calcium ion is required for this esterolysis. This reaction involves the formation of a trimethylacetyl enzyme complex which has been shown by two procedures, burst in p-nitrophenol liberation and incorporation of 14C-trimethylacetyl, to have one trimethylacetyl group per molecule of protein. A trimethylacetyl enzyme complex containing 0.9 eq of trimethylacetyl has been isolated. Incorporation of trimethylacetyl is reflected in a concomitant loss in activity toward p-nitrophenyl acetate and for hydroxylamine incorporation into carbobenzoxy-l-glutaminylglycine. Studies on the inhibition of transglutaminase activity by p-nitrophenyl trimethylacetate and formation of 14C-trimethylacetyl enzyme, both as a function of calcium ion concentration, indicate that the same divalent cation-enzyme complexes are involved in p-nitrophenyl acetate hydrolysis and in p-nitrophenyl trimethylacetate hydrolysis. Thus the apparent dissociation constants for the calcium-transglutaminase complex, as determined by inhibition and by trimethylacetyl enzyme formation at pH 7.0, are 0.37 ± 0.2 x 10-3 m and 0.54 ± 0.2 x 10-3 m, respectively. These values are comparable to the reported activator constant of calcium, 0.4 ± 0.2 x 10-3 m at pH 7.0, determined from the kinetics of p-nitrophenyl acetate hydrolysis. A single radioactive peptide has been separated from a proteolytic digest of 14C-trimethylacetyl transglutaminase. Studies on the isolated peptide indicate that the trimethylacetyl group is bound in thioester linkage to a cysteine —SH group and to the same —SH group in the enzyme with which iodoacetamide reacts to form the inactive carbamidomethyl enzyme. These results are presented in support of a previously suggested cation-controlled mechanism of enzyme action involving the intermediate formation of an acyl enzyme through thioester linkage.