Thrombin-like enzyme from Lachesis muta muta venom: isolation and topographical analysis of its active site structure by means of the binding of amidines and guanidines as competitive inhibitors

Abstract A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62127 (M × cm) −1 . The enzyme hydrolysed Bz-Arg-Nan with K s = 0.233 + 0.08 mM and k cat = 2.80 ± 0.07 sec −1 . All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with K i values in the range 6.2 PM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analysed in terms of the binding of the inhibitors with the subsite S 1 the specificity pocket of the enzyme. K i values were discussed in accordance with those for trypsin inhibition. β-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the K i values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log 1 K i and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hiickel molecular orbital theory. A model for the binding of p -benzamidine derivatives with the primary specificity S 1 subsite of the enzyme active site was proposed.