Abstract
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.
Highlights
In recent years considerable research efforts have been expended in the design and synthesis of glycosidase inhibitors to understand about the active site structures and mechanisms of these interesting enzymes and in generating new therapeutic agents
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
Initial kinetic assessments revealed that pepstatin A is a competitive inhibitor of Xyl I with an IC50 value of 3.6 Ϯ 0.5 M (Fig. 1)
Summary
Materials—Oat spelled xylan, dinitrosalicylic acid, pepstatin A, and o-phthalaldehyde (OPTA) were obtained from Sigma. Producing Xyl I is an alkalothermophilic actinomycete having optimum growth at pH 9 and 50 °C. One unit of xylanase activity was defined as the amount of enzyme that produced 1 mol of xylose equivalent per min using oatspelled xylan as the substrate under assay conditions. For the Lineweaver-Burk analysis Xyl I (2 M) was incubated with pepstatin A at (1 M) and (2 M) and assayed at increased concentrations of xylan (1–10 mg/ml) at 50 °C for 30 min. In Dixon’s method [30], xylanolytic activity of Xyl I (2 M) was measured in the presence of 5 and 10 mg/ml xylan at concentrations of pepstatin A ranging from 0 to 7 M at 50 °C for 30 min.
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