Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: Correlation of the kinetic parameters with the inhibitor induced conformational changes

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechan ism against the aspartic proteases, pepsin and F-prot from Aspergillus sait oi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-t ight binding inhibition: E + I reversible arrow (k(3), k(4)) EI reversible arrow (k(5), k(6)) EI*. The K-i values for the first reversible complex (EI ) of ATBI with pepsin and F-prot were (17 +/- 0.5) X 10(-9) M and (3.2 +/- 0.6) X 10(-6) M, whereas the overall inhibition constant K-i* values were ( 55 +/- 0.5) X 10(-12) M and (5.2 +/- 0.6) X 10(-8) M, respectively. The rat e constant k5 revealed a faster isomerization of EI for F-prot [(2.3 +/- 0. 4) X 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) X 10(-4) s(-1)]. However, ATB I dissociated from the tight enzyme-inhibitor complex (EI*) of F-prot faste r [(3.8 +/- 0.5) X 10(-1) s(-1)] than pepsin [(2.5 +/- 0.4) X 10(-6) s(-1)] . Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The bin ding of the inhibitor with the aspartic proteases and the subsequent confor mational changes induced were monitored by exploiting the intrinsic tryptop hanyl fluorescence. The rate constants derived from the fluorescence data w ere in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of El to EI*. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inacti vation.