A new approach in the active site investigation of an inverting β-D-xylosidase from Thermobifida fusca TM51.

Csaba Attila Fekete, László Kiss

The protein journal 2013 Feb

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The catalytic amino acid residues of the β-D-xylosidase (EC 3.2.1.37; GH43), from Thermobifida fusca TM51 (TfBXyl43), were investigated by direct chemical modifications. The pH dependence curves of the kinetic parameters (k(cat) and k(cat)/K(M)) gave pK values for the free enzyme (5.55 ± 0.19; 6.44 ± 0.19), and pK values of for the enzyme-substrate complex (4.85 ± 0.23; 7.60 ± 0.28) respectively, by using an artificial substrate p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl). The detailed inhibition studies demonstrated well the hydrophobic character of the glycon binding site. Carbodiimide-mediated chemical modifications of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in the presence of glycine methyl ester supports the conclusion that a carboxylate residue can be fundamental in the catalytic process. We have also synthesized and tested N-bromoacetyl-β-D-xylopyranosylamine (NBAXA) for TfBXyl43 as an affinity label, which also inactivated the enzyme irreversible. The pH dependence studies in both cases of inactivation revealed that the modified group is the catalytic proton donor (NBAXA pK(A) = 6.68 ± 0,1; EDAC pK(A) = 7.42 ± 0.22) which displays its essential role in the hydrolytic process. The β-D-xylopyranosylazide as competitive inhibitor protected the enzyme in all cases against the inactivation, suggesting that the chemical modification which has an impact on the activity took place in the active center. Changing of the enzyme conformation was followed by CD spectroscopy, as a result of the NBAXA inactivation. Our study is important because to our knowledge no similar investigations were made in the case of an inverting β-D-xylosidase.

Citation

Csaba Attila Fekete, László Kiss. A new approach in the active site investigation of an inverting β-D-xylosidase from Thermobifida fusca TM51. The protein journal. 2013 Feb;32(2):97-105