Journal of Fluorescence

, Volume 23, Issue 2, pp 311–321 | Cite as

Mechanistic Insights into the Inhibition of Endo-β 1,4 Xyloglucan Hydrolase by a Classical Aspartic Protease Inhibitor



This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC 50 and K i values of 3.5 ± 0.5 μM and 1.25 ± 0.5 μM respectively. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 14.5 ± 1.5 × 10−5 s−1 and 2.85 ± 1.2 × 10−8 s−1 respectively, whereas the overall inhibition constant K i * was 27 ± 1 nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase 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.


Endo-β 1,4 xyloglucan hydrolase Pepstatin Enzyme kinetics Slow-tight binding Inactivation mechanism 



MR and VM acknowledge the financial support and the senior research fellowship from CSIR Emeritus Scheme, Govt. of India respectively. MR is thankful to Dr Barry McCleary, Megazyme International Ireland Ltd for xyloglucan from tamarind seeds.


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Division of Biochemical SciencesNational Chemical LaboratoryPuneIndia

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