Mechanism of Action of Guinea Pig Liver Transglutaminase

Abstract

Abstract Evidence is presented that transglutaminase catalyzes the hydrolysis of p-nitrophenyl acetate and that this esterolysis occurs at or near a site involved in the other activities of the enzyme. Calcium or strontium is required for this reaction. p-Nitrophenyl acetate hydrolysis is effectively inhibited by another transglutaminase substrate, carbobenzoxy (CBZ)-l-glutaminylglycine. Parallel losses in the esterolytic and hydroxamate incorporation activities are observed following treatment of transglutaminase with iodoacetamide. The pH profile for esterolysis is similar, in certain respects, to that for hydroxamate incorporation. The turnover number, 92 ± 13 moles of p-nitrophenol formed min-1 mole of enzyme-1 at pH 6, 24°, in 5% isopropyl alcohol and at saturating levels of calcium ion, is approximately 20- and 100-fold those at pH 8 reported for rabbit muscle 3-phosphoglyceraldehyde dehydrogenase and chymotrypsin, respectively. A kinetic evaluation of this calcium-dependent transglutaminase-catalyzed esterolysis reaction shows that the mechanism differs from that of the hydroxamate incorporation reaction. The activator constant (Ka) of calcium (0.8 ± 0.25 x 10-3 m at pH 6, 24°) and the apparent dissociation constant (K'd) determined for the metal-enzyme complex by equilibrium dialysis (1 ± 0.6 x 10-3 m at pH 7, 2°) are approximately an order of magnitude lower than the Ka of calcium reported for the hydroxamate incorporation reaction and the Kd values derived from calcium-induced conformational changes in the enzyme protein. The Ka of calcium in the esterolysis reaction shows a marked decrease as the pH is increased with an apparent pK at 6.4, while the Kd for this cation, as measured by ultraviolet difference spectrophotometry, is independent of pH over the range studied. The calcium ion level required for reactivity of an essential —SH group of transglutaminase was found to be in the range of the Ka of calcium in the esterase reaction and the K'd for the calcium-enzyme complex as measured by equilibrium dialysis. Re-examination of the hydroxamate incorporation reaction at pH 5.4 and at high substrate and low calcium ion levels revealed a dependence of maximum velocity on cation concentration and a nonlinear relationship between reciprocal velocity and reciprocal metal ion concentration. These observations support and extend a previous suggestion that transglutaminase functions in combination with divalent cation as a metal-enzyme complex. They also form the basis for a proposition that metal ion combines in two different positions on the enzyme, thus giving rise to three possible enzyme-metal complexes. A complex containing only 1 metal ion serves to catalyze the hydrolysis of p-nitrophenyl acetate, whereas the complex in which cation is bound at both sites in the enzyme is essential for amine incorporation into the glutamine substrate.