Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.
1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.
D000577:Amides / Q000138:chemical synthesis*; D000579:Amidinotransferases; D000588:Amines; D001665:Binding Sites; D002103:Cadaverine; D002118:Calcium; D002250:Carbon Radioisotopes; D003619:Dansyl Compounds; D005176:Factor XIII / Q000134:cerebrospinal fluid; D005453:Fluorescence; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D010666:Phenylpropionates; D013438:Sulfhydryl Compounds; D013917:Thrombin; D005723:gamma-Glutamyltransferase
D000577:Amides; D000588:Amines; D002250:Carbon Radioisotopes; D003619:Dansyl Compounds; D010666:Phenylpropionates; D013438:Sulfhydryl Compounds; D005176:Factor XIII; D000579:Amidinotransferases; D005723:gamma-Glutamyltransferase; D013917:Thrombin; D002103:Cadaverine; D002118:Calcium