Mechanistic Insights into the Inhibition of Endo-β 1,4 Xyloglucan Hydrolase by a Classical Aspartic Protease Inhibitor
- 243 Downloads
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.
KeywordsEndo-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.
- 10.Yoshizawa T, Shimizu T, Yamabe M, Taichi M, Nishiuchi Y, Shichijo N et al (2011) Crystal structure of basic 7S globulin, a xyloglucanspecific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase. FEBS J 278:1944–1954PubMedCrossRefGoogle Scholar
- 11.Yoshizawa T, Shimizu T, Hirano H, Sato M, Hashimoto H (2012) Structural basis for the inhibition of xyloglucan-specific endo-β-1,4-glucanase (XEG) by XEG-protein inhibitor. J Biol Chem. doi: 10.1074/jbc.M112.350520
- 21.Cleland WW (1979) The kinetics of enzyme catalyzed reactions with two or more substrates or products—II: inhibition, nomenclature and theory. Biochim Biophys Acta 67:173–187Google Scholar
- 29.Stutz AE (1999) Immunosugars as glycosidase inhibitors: nojirimycin and beyond. Wiley, Weinheim, pp 95–120Google Scholar
- 31.Cheung HC (1991) Topics in fluorescence spectroscopy. In: Lakowicz JR (ed) Principles, vol 2. Plenum, New York, pp 127–176Google Scholar