<Originals>Trypsin-Catalyzed Hydrolysis of Synthetic Substrate, Tosyl-D-Arginine Methyl Ester

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概要

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• Trypsin-catalyzed hydrolysis of a synthetic ester substrate, tosyl-D-arginine methyl ester (D-TAME), whose L-enantiomer is widely used as a substrate for trypsin, was carried out and followed by the entire progression curve method.^1 This ester substrate which is a D-enantiomer of arginine, is hydrolyzed to nearly 100% product. The experiments were performed by changing pH and the initial substrate concentration. The double reciprocal plot of the velocity of hydrolysis against the residual substrate concentration gives a good linearity. The straight lines below pH 8 in this plot intersect at one point. At a constant pH the slope of double reciprocal plot increases with the increase of the initial substrate concentration. We postulated an enzymatic reaction mechanism^2 like the ordered Bi-Bi reaction mechanism proposed by Cleland.^3 The mechanism includes not only the action of substrate on the active site but also the action of allosteric modifier (termed as "functionant") at the second site of trypsin which is known as an allosteric site (termed as "functional site") for the substrate activation.^4 Each binding and releasing phase at the active or the functional site results in a conformational change of the enzyme molecule. The data obtained were analyzed according to the corresponding rate equation and thus various parameters such as the dissociation constants for enzyme-substrate and enzyme-functionant complexes and the rates of subsequent conformational changes in the enzyme molecule were determined. The rates of conformational change vary in exponential form with changing pH. The pK value of the rate constant concerned with the active site is 7.2 and that of the rate constant concerned with the functional site is 8.4.

• 近畿大学の論文
• 1979-09-28

著者

• Goto Sachio

  Department Of Biophysical Chemistry Faculty Of Agriculture Kinki University

• Goto Sachio

  Department Of Biochemistry Kinki University School Of Medicine

関連論文

• Trypsin-Catalyzed Hydrolysis of Synthetic Substrate, Tosyl-D-Arginine Methyl Ester
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