Requirements for Stereospecificity in Hydrolysis by Chymotrypsin: Diethyl β-Acetamidoglutarate

Abstract

AS part of our study of the relation of structure of substrate to stereospecificity in enzyme reactions¹, we wish to report asymmetric hydrolysis of diethyl β-acetamidoglutarate by α-chymotrypsin. This compound was prepared by acetylation of diethyl β-aminoglutarate², b.p. 142–144°/2 mm.; anal. calc. for C₁₁H₁₉NO₅: C, 53.88; H, 7.81; N, 5.71. Found: C, 54.33; H, 7.84; N, 5.50. (Chymotrypsin was from Worthington, salt free.) A solution of 0.544 gm. of the ester and 0.524 gm. of the enzyme in 52.4 ml. of 0.02 M phosphate buffer was allowed to react at 27° in a pH-stat at pH 7.8, one ester group being 80–85 per cent hydrolysed in 5 hr. The reaction showed reproducible first-order kinetics, k ₁ = 0.0057 min.⁻¹, indicating extremely weak catalytic activity. However, the reaction was stereospecific. Neutralization of the alkali which had been added, evaporation to dryness and extraction with acetone led to optically active monoethyl-β-acetamidoglutarate, 0.318 gm., 78 per cent yield, m.p. 93–94 per cent. [α]²⁵⁰ _D + 5.9° (5 per cent acetone)³. Anal. calc. for C₉H₁₅NO₅: C, 49.73; H, 6.96; N, 6.45. Found: C, 49.80; H, 6.92 ; N, 6.51. The half-ester was prepared in the racemic form by hydrolysis of the di-ester to the di-acid, β-acetamidoglutaric acid, m.p. 185–186° dec., anal. calc. for C₇H₁₁NO₅: N, 7.41; found: N, 7.38; conversion of the di-acid to β-acetamidoglutaric anhydride, m.p. 140–141°; anal. calc. for C₇H₉NO₄: C, 49.11; H, 5.30; N, 8.18; found: C, 49.05; H, 5.26; N, 8.16; and conversion of the anhydride to racemic monoethyl β-acetamidoglutarate, m.p. 77.78°. Anal. found: C, 49.71; H, 6.92; N, 6.42. The racemic and (+) monoesters had identical infra-red spectra in chloroform.