Open AccessThe Kinetics of Hydrolysis of some Extended N‐Aminoacyl‐L‐lysine Methyl Esters by Bovine α and β ‐Trypsins
Author(s) -
GREEN George D. J.,
TOMALIN Geoffre
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10771.x
Subject(s) - trypsin , chemistry , enzyme kinetics , hydrolysis , lysine , reaction rate constant , stereochemistry , enzyme , residue (chemistry) , nucleophile , kinetics , medicinal chemistry , organic chemistry , active site , amino acid , catalysis , biochemistry , physics , quantum mechanics
1 The action of two active forms of bovine trypsin (α and β‐trypsin) on a series of specific methyl ester substrates of general formula: N‐acetyl‐(glycyl) n ‐L‐lysine methyl ester (n = 0, 1, 2) and N 2 ‐benzoyl‐L‐arginine ethyl ester have been investigated. With the L‐lysine methyl esters the catalytic rate constant for hydrolysis (k eat ,) was found to be significantly lower for α‐trypsin than for β‐trypsin, whereas with N 2 ‐benzoyl‐L‐arginine ethyl ester there was no significant difference for the two enzymes. 2 By measurement of the kinetic constants (k eat , and K m ) in the presence of a nucleophile, which competes with water in the deacylation process, it has been shown that, in common with the specific ester substrates of trypsin, the rate‐determining step for the extended L‐lysine methyl esters is also deacylation of the enzyme. 3 It has been found that by extending the aminoacyl group of N‐acetyl‐ l ‐lysine methyl ester by one glycine residue (n = 1), a greatly enhanced deacylation rate constant is observed for both a and β‐trypsin. The higher rate constants were maintained at the higher level by the addition of a further glycine residue (n = 2). These results have been interpreted in terms of the ‘induced fit’ hypothesis the substrates binding to an enzyme subsite adjacent to the active site. 4 The β‐trypsin‐catalysed hydrolysis of the L‐lysine substrates was investigated over a range of temperature (15–35 °C). The Arrhenius law was obeyed, within experimental error, by all three substrates allowing the estimation of the thermodynamic function of activation (ΔS* and ΔH*) for the deacylation reactions. The significantly higher values of ΔS* and ΔH* obtained for the two extended substrates are interpreted in terms of additional hydrogen bonding between the longer aminoacyl chains and the enzyme molecule. The results are compared with those for non‐extended specific substrates, which have a possible hydrophobic interaction with the enzyme surface.