Abstract
Effects of hydrogen-bonding interactions of amide groups on reactivity of esters to alpha-chymotrypsin were studied. Of the methyl esters studied, only that from acetyl-L-phenylalanine has k3 rate-limiting. In methyl beta-phenylpropionates an alpha-acetamido substituent increased k2 greater than 550 times, k3 approximately 5 times; an alpha-acetylclycyloxy substituent increased k2 approximately 2 times, k3 approximately 6 times, both in comparison with the alpha-acetoxy esters. Essentially all carboxamidomethyl esters studied have k3 rate-limiting; reactivity to hydroxide is only 4 times that of methyl esters. In alpha-substituted beta-phenylpropionates, carboxamido-methyl esters show k2 values greater than 110 times greater than 280 times, greater than 26 times, and 7 times the k2 values of the methyl esters for the alpha substituents, acetoxy, acetylglycyloxy, hydroxy, and hydrogen, respectively. In esters of alpha-acetamido acids, carboxamidomethyl esters show k2 values 44 times, greater than 110 times, greater than 12 times, and approximately 33 times the k2 values of the methyl esters of glycine, alanine, leucine, and phenylalanine, respectively. Cyanomethyl esters also had k3 rate-limiting. Hydrogen-bonding to the enzyme of either an alpha-acetamido group or a carboxamidomethyl group combined with bonding of the beta-aryl group, orients the hydrolyzing groups properly, increasing k2. Hydrogen-bonding of both alpha-acetamido and carboxamido-methyl groups is effective to a lesser degree. The amide group appears to have small effects on Ks as hydrogen bonding is balanced by desolvation. It is proposed that desolvation during bonding increases k2 and Ks.
Highlights
CO&H, compound I-N, the fl-phenyl group fits into the primary binding subsite, a pocket made by the chain of residues 189 to 194 on one side and 214 to 220 on the other; the a-acylamido group has its N-H hydrogen directed to >C=O of Ser 214 and its >C=O toward the side chain of Met 192; the hydrolyzing
Hydrolysis by cu-chymotrypsin of the carboxamidomethyl esters of m-N-acetylalanine, oL-N-acetylleucine and DL-N-acetylphenylalanine stopped at 50% reaction; the cY-acetamido compounds show high L-stereospecificity
The p-nitrophenyl esters were studied titrimetrically at pH 7.8, under the same conditions as the other esters, rather than spectrophotometrically with buffer, as is usually done. Both the acid and p-nitrophenol produced in the hydrolysis consumed alkali, and correction for this was applied
Summary
2-Hydrory-3.phenylpropionate; VII-m-@-Phenyllactic 41 g of a-chloracetamide acid The solution was washed, dried, and concentrated and the residue was treated with ethyl acetate/petroleum ether, leading to XVIII, m.p. 102-103”, [a]: -65” (C, 1.3 in chloroform); literature value [23] m.p. 103-104”, [a]:’ -66.8”. The thick reaction mixture was diluted with ethyl acetate and filtered Both filtrate and residue contained product, which was obtained after much fractional crystallization from ethanol petroleum ether and from ethyl acetate, m.p. 120-121”, [a]: c2.9” (C, 2.4 in ethanol), literature value [24] m.p. 120.5-121.5”, [a]:: +2.2”. Stock solutions of substrates were prepared in acetonitrile for compounds which did not have a-acetamido groups, and in water for the a-acetamido compounds Kinetic studies of the former were carried out in 10%.
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