Inhibition of 1,4-β-d-Xylan Xylanohydrolase by the Specific Aspartic Protease Inhibitor Pepstatin

Vinod Vathipadiekal, Mala Rao
2004 Journal of Biological Chemistry  
This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC 50 value 3.6 ؎ 0.5 M. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to
more » ... e second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 ؎ 1 ؋ 10 ؊5 and 3.0 ؎ 1 ؋ 10 ؊8 s ؊1 , respectively. The K i value for the formation of EI complex was 1.5 ؎ 0.5 M, whereas the overall inhibition constant K i * was 28.0 ؎ 1 nM. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.
doi:10.1074/jbc.m407866200 pmid:15317808 fatcat:xvblurzbavgf3obxdrsqm5tx64