Kinetics of Hydrolysis of Amide and Anilide Substrates of p-Guanidino-L-Phenylalanine by Bovine and Porcine Trypsins

Abstract

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

“…Moreover, aryl amides should be easier to cleave than aliphatic amides from a purely electronic point of view, due to the less pronounced double bond character of an amide bond adjacent to an aromatic ring system. This situation can also result in faster reactions catalysed by enzymes involving acyl‐enzyme intermediates such as the serine protease trypsin, for which nitroanilide substrates exhibited greater performance constants than the corresponding primary amides . However, results comparable to those reported here were also obtained from studies on chromogenic and fluorogenic acyl donor substrates for factor XIIIa.…”

Formation of a Trypsin−Borate−4-Aminobutanol Ternary Complex

“…Kinetic analysis of initial rate data as a function of the substrate and borate or 4-aminobutanol yielded the following values: K S ) 3.47 mM, K B ) 80 mM, and K I ) 3.5 mM. This K S value is somewhat higher than the reported value of 1.08 mM obtained under slightly different conditions (14). Direct fitting of eq 1 using Mathematica yielded an R of 0.2.…”

“…Although a conclusive interpretation of the 1 H shits is not possible at this point, the above analysis indicates that the ternary complex involves a borate-Ser 195 bond, either exclusively (Scheme 1A) or, less probably, as a component of a mixture of species (Scheme 1). Spectrophotometric assays of trypsin were performed at pH 8.0 in a 100 mM HEPES buffer using Ac-Arg-pNA as the substrate (14). The resulting data were intrepreted using the equation for cooperative binding of two ligands, I (4aminobutanol) and B (borate), both of which are competitive with substrate binding (26).…”

“…The boron spectra were obtained at 25 °C using 5 mm quartz NMR tubes (Wilmad, Buena, NJ), and using a 5 mm Nalorac probe (Martinez, CA), modified to reduce the boron background. Spectrophotometric assays were performed on a Beckman DU 640 spectrophotometer using 0.3 mM acetylarginine p -nitroanilide (Ac-Arg-pNA) as a chromogenic substrate and 200 nM porcine pancreatic trypsin and measuring the absorbance at 410 nm ( , ). Kinetic data were analyzed using Mathematica (Wolfram Research, Champaign, IL).…”

Physiological perturbations in juvenile cuttlefish Sepia officinalis induced by subchronic exposure to dissolved zinc

“…Trypsin activity was measured according to Tsunematsu et al (1985) using 1 mM Na-benzoyl-Arg-p-nitroanilide as substrate in a 0.1 M Tris buffer at pH 9. Briefly, 20 ll of supernatant (or extraction Tris buffer pH 8 for blank) were mixed to 100 ll of substrate, before incubation for 1 h at 25°C.…”