1. Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin
Y Hatanaka, H Tsunematsu, K Mizusaki, S Makisumi Biochim Biophys Acta. 1985 Dec 20;832(3):274-9. doi: 10.1016/0167-4838(85)90260-2.
The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.
2. Characterization of active derivatives produced by acetamidination and selective autolysis of bovine trypsin
T Kumazaki, S Ishii J Biochem. 1979 Feb;85(2):581-90. doi: 10.1093/oxfordjournals.jbchem.a132367.
A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated alpha- and amidinated beta-trypsins. The latter component (Am-beta-trypsin), which consisted of a single polypeptide chain, was allowed to autolyze at pH 7.8, 25 degrees C for 3.5 h and a new active component named Am-delta-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-delta-trypsin was derived by primary cleavage of the bond Arg105-Val106. Cleavage at Arg55-Leu56, on the other hand, appeared to lead to inactivation of Am-beta-trypsin. The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-delta-, Am-beta-, and Am-alpha-trypsins. Am-delta-trypsin resembled Am-beta-trypsin in these properties, but did not resemble Am-alpha-trypsin which had a cleavage at Lys131-Ser132.
3. Interactions of derivatives of guanidinophenylglycine and guanidinophenylalanine with trypsin and related enzymes
H Tsunematsu, S Makisumi J Biochem. 1980 Dec;88(6):1773-83. doi: 10.1093/oxfordjournals.jbchem.a133152.
Ethyl N-benzoyl-p- and m-guanidino-DL-phenylglycinates (DL-Bz-p-GPG-OEt and DL-Bz-m-GPG-OEt), and ethyl N-benzoyl-p-guanidino-L- and D-phenylalaninates (L-Bz-p-GPA-OEt and D-Bz-p-GPA-OEt) were synthesized. The ester of the racemic p-guanidinophenylglycine derivative was completely hydrolyzed by trypsin, pronase, alpha-chymotrypsin, and thrombin, though hydrolysis by the latter two enzymes was much slower. Papain hydrolyzed this ester substrate stereospecifically at a moderate rate and left the ester derivative of the D-enantiomer unaltered. Optical resolution of DL-Bz-p-GPG-OEt with papain gave N-benzoyl-p-guanidino-L-phenylglycine (L-Bz-p-GPG-OH) and the ester of the D-enantiomer of this amino acid derivative. On the other hand, DL-Bz-m-GPG-OEt was completely hydrolyzed by pronase and was stereospecifically hydrolyzed by papain, but was unaffected by trypsin, alpha-chymotrypsin, and thrombin. The trypsin-catalyzed hydrolysis of N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-pNA) was inhibited competitively by this ester. The specificity constant (kcat/Km) for L-Bz-p-GPG-OEt was about 57 times smaller than that for a specific ester substrate, ethyl N alpha-benzoyl-L-argininate (LO-Bz-Arg-OEt), while the value for the D-enantiomer of the former is about 14 times larger than that for the D-enantiomer of the latter. L-Bz-p-GPA-OEt has a specificity constant comparable to that for L-Bz-Arg-OEt. The value for the former is about 51 times larger than that for L-Bz-p-GPG-OEt. This suggests that the existence of the beta-methylene group in L-Bz-p-GPA-OEt is important in relation to the higher susceptibility of the ester to trypsin-catalyzed hydrolysis. In contrast with the L-enantiomer, D-Bz-p-GPA-OEt was found to be as competitive inhibitor for the hydrolysis of L-Bz-Arg-pNA. A significant difference was found between the stereospecificities of hydrolysis of the ester substrates of the two amino acid derivatives by trypsin.
