|Title||Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: Histidine characterization by NMR spectroscopy|
|Publication Type||Journal Article|
|Year of Publication||2007|
|Authors||Schubert, M, Poon, DKY, Wicki, J, Tarling, CA, Kwan, EM, Nielsen, JE, Withers, SG, McIntosh, LP|
We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile H-1(delta 1) and H-1(epsilon 1) atoms of four histidines (t(1/2) similar to 0.1-300 s at 30 degrees C and pH similar to 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pK(a) values and neutral tautomeric forms were determined from their pH-dependent C-13(epsilon 1)-H-1(epsilon 1) chemical shifts, combined with multiple-bond H-1(delta 2/epsilon 1)-N-15(delta 1/epsilon 2) scalar coupling patterns. Remarkably, these pK(a) values span more than 8 log units such that at the pH optimum of similar to 6 for Cex activity, His107 and His205 are positively charged (pK(a) > 10.4), His85 is neutral (pK(a) < 2.8), and both His80 (pK(a) = 7.9) and His114 (pK(a) = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pK(a) value of His80 drops from 7.9 to < 2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.
|URL||<Go to ISI>://000247340300007|