Cryoenzymology of trypsin. A detailed kinetic study of the trypsin-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at low temperatures.

J P Malthouse, A I Scott

Index: Biochem. J. 215(3) , 555-63, (1983)

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Abstract

A detailed study of the kinetics of the trypsin (EC 3.4.21.4)-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester in cryosolvents at 0 degrees C and below was undertaken. The pH-dependences of kcat, Km, k+2, k+3 and Ks were determined under cryoenzymological conditions and are compared with previous results [Antonini & Ascenzi (1981) J. Biol. Chem. 256, 12449-12455] obtained in fully aqueous media at ambient temperatures. Below pH 5.0 the kinetics, and presumably the mechanism of catalysis, are not significantly perturbed under cryoenzymological conditions. However, it is shown that below pH 5.0 both Km and Ks are decreased under these conditions but that both are increased at pH 6.7 relative to the results obtained in fully aqueous media at ambient temperatures. The effects of the cryoenzymological conditions on the individual catalytic parameters are discussed. The acylation rate constant, k+2, is essentially constant at pH 4.2 and 5.0 but decreases at lower pH values with an apparent pKa of approx. 4.0. In view of the low enthalpy of ionization associated with this pKa it is suggested that this group is the carboxy group of aspartic acid-189, which binds the positively charged lysine side chain of the substrate. The mechanistic implications of the results for the acylation step are discussed. It is also shown that only at low pH values can significant amounts of acylated trypsin be accumulated.