Mechanism of Action of Guinea Pig Liver Transglutaminase

Abstract

Abstract The activator constant, Ka, of calcium in the transglutaminase reaction has been estimated from kinetic studies as 6.9 ± 1.5 x 10-3 m at pH 6 in 0.1 m tris(hydroxymethyl) aminomethane-acetate, 25°. The dissociation constant, Kd, for the calcium-transglutaminase complex, determined by two direct binding experiments, was found to be 8 ± 2 x 10-3 m at pH 6 in 0.1 m Tris-acetate containing 0.5 m NaCl, 25° (ultraviolet difference spectrophotometry), and 7.9 ± 1.5 x 10-3 m at pH 6 in 0.1 m Tris-acetate, 25° (protection against irreversible inhibition). The Km for carbobenzoxy-l-glutaminylglycine with the fully activated calcium enzyme and the maximum turnover number with this substrate were found to be 21.7 ±4 x 10-3 m and 3600 ± 300 min-1, respectively. The Ka of strontium and the Kd (difference spectrophotometry) for the strontium-transglutaminase complex have been determined as 80 ± 20 x 10-3m and 90 ± 25 x 10-3m, respectively. The maximum turnover number for carbobenzoxy-l-glutaminylglycine is essentially that observed for the calcium-activated enzyme, while the Km for this substrate with the fully strontium-activated enzyme was found to be 75 ± 10 x 10-3 m. Carbobenzoxy-l-phenylalanine inhibits transglutaminase in a reversible competitive manner. The apparent inhibitor constant for this compound is a function of the calcium concentration at less than saturating levels of metal ion. The inhibitor constant, Ki, for carbobenzoxy-l-phenylalanine with the fully activated calcium enzyme was estimated as 8 ± 3 x 10-3 m. At less than saturating levels of calcium ion, the degree of protection against irreversible inhibition of transglutaminase by 5,5'-dithiobis(2-nitrobenzoic acid) was observed to be a function of carbobenzoxy-l-phenylalanine concentration. No protection by carbobenzoxy-l-phenylalanine against this irreversible inhibitor was observed in the absence of activating metal ion. The reversible competitive inhibition afforded by chloracetic acid was characterized by the same observed Ki value at two low levels of calcium ion. This competitive inhibitor showed no protection against irreversible enzyme inhibition either in the presence or absence of calcium ion. These facts are consistent with a mechanism in which transglutaminase functions in combination with the divalent cation as a metal-enzyme complex. The results also suggest that a conformational change in the enzyme protein occurs upon combination with activator metal.