Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism.
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 IC50 value 3.6 +/- 0.5 microm. 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 the 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 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* 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.
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411-008, India.
Vinod
V
Vathipadiekal
Mala
M
Rao
D039903:Actinobacteria / Q000201:enzymology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010436:Pepstatins / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D004791:Enzyme Inhibitors; D010436:Pepstatins; D011480:Protease Inhibitors; D006639:Histidine; D043364:Endo-1,4-beta Xylanases; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin