Tsunematsu H, Makisumi S
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.
合成了N-苯甲酰基-p-和m-胍基-DL-苯甘氨酸乙酯（DL-Bz-p-GPG-OEt和DL-Bz-m-GPG-OEt）以及N-苯甲酰基-p-胍基-L-和D-苯丙氨酸乙酯（L-Bz-p-GPA-OEt和D-Bz-p-GPA-OEt）。外消旋p-胍基苯甘氨酸衍生物的酯可被胰蛋白酶、链霉蛋白酶、α-胰凝乳蛋白酶和凝血酶完全水解，不过后两种酶的水解速度要慢得多。木瓜蛋白酶以中等速度立体特异性地水解该酯底物，而D-对映体的酯衍生物则保持不变。用木瓜蛋白酶对DL-Bz-p-GPG-OEt进行光学拆分，得到N-苯甲酰基-p-胍基-L-苯甘氨酸（L-Bz-p-GPG-OH）和该氨基酸衍生物D-对映体的酯。另一方面，DL-Bz-m-GPG-OEt可被链霉蛋白酶完全水解，并被木瓜蛋白酶立体特异性水解，但不受胰蛋白酶、α-胰凝乳蛋白酶和凝血酶的影响。该酯对胰蛋白酶催化的Nα-苯甲酰基-L-精氨酸对硝基苯胺（L-Bz-Arg-pNA）水解有竞争性抑制作用。L-Bz-p-GPG-OEt的特异性常数（kcat/Km）比特定酯底物Nα-苯甲酰基-L-精氨酸乙酯（LO-Bz-Arg-OEt）的特异性常数小约57倍，而前者的D-对映体的值比后者的D-对映体的值大约14倍。L-Bz-p-GPA-OEt的特异性常数与L-Bz-Arg-OEt的相当。前者的值比L-Bz-p-GPG-OEt的值大约51倍。这表明L-Bz-p-GPA-OEt中β-亚甲基的存在对于该酯对胰蛋白酶催化水解的更高敏感性很重要。与L-对映体相反，发现D-Bz-p-GPA-OEt是L-Bz-Arg-pNA水解的竞争性抑制剂。在胰蛋白酶对两种氨基酸衍生物酯底物的水解立体特异性之间发现了显著差异。
