Involvement of a lysine residue in the active site of a thermostable xylanase from Thermomonospora sp.
A highly thermostable xylanase (Xyl I) produced by Thermomonospora sp. was purified to homogeneity and was classified as a family 10 xylanase based on its molecular weight (38,000 Da) and isoelectric point (4.1). K2d analysis showed that the secondary structure of Xyl I was made up of 38% alpha-helix and 10% beta-sheet. The optimal temperature for the activity of Xyl I was 80 degrees C. Xyl I was highly thermostable with half-lives of 86, 30, and 15 min at 80, 90, and 100 degrees C respectively. Xyl I was stable in an expansive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemoaffinity labeling and trinitrobenzenesulphonic acid (TNBS) as chemical modifier have revealed the presence of a single lysine residue in the active site of Xyl I. The high pK value for the basic limb of the pH profile reflects the ionization of a lysine residue. The higher K(m) values and similar k(cat) values of the TNBS modified enzyme in comparison to native enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site.
George
S P
SP
Division of Biochemical Sciences, Pune, 411 008, India.
Ahmad
A
A
Rao
M B
MB
D000192:Actinomycetales / Q000201:enzymology*; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D017433:Protein Structure, Secondary; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009764:o-Phthalaldehyde / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009764:o-Phthalaldehyde; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D008239:Lysine