HYDROLYSIS OF ARTIFICIAL SUBSTRATES BY ENTEROKINASE AND TRYPSIN AND THE DEVELOPMENT OF A SENSITIVE SPECIFIC ASSAY FOR ENTEROKINASE IN SERUM

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates α-N-Benzoyl-dl-arginine ethyl ester HCl, α-N-Benzoyl-dl-arginine-p-nitroanilide HCl and α-N-Benzoyl-dl-arginine-2-napthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-l-aspartyl-l-lysyl-2-naphthylamide (Gly-(Asp)4-Lys-Nap) by these proteases was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response of CA2+ was sigmoidal party due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+. © 1979.