Mechanism of Action of Guinea Pig Liver Transglutaminase

作者：J.E. Folk, P.W. Cole, John P. Mullooly · 发表于：Journal of Biological Chemistry · 年份：1967 · DOI：10.1016/s0021-9258(18)99616-x · 被引用次数：46 · 研究领域：Blood properties and coagulation、Erythropoietin and Anemia Treatment、Enzyme function and inhibition

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 calc...