Interactions of derivatives of guanidinophenylglycine and guanidinophenylalanine with trypsin and related enzymes.
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.
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006146:Guanidines / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010206:Papain / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
D006146:Guanidines; C028772:guanidinophenylglycine; C017351:4-guanidinophenylalanine; D010649:Phenylalanine; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin; D010206:Papain; D011402:Pronase; D005998:Glycine