Enzymatic degradation of κ-carrageenan in aqueous solution.
Enzymatic degradation of standard κ-carrageenan and the low-gelling hybrid κ-/μ-carrageenan were conducted using recombinant Pseudoalteromonas carrageenovora κ-carrageenase. The initial velocity of the enzyme was determined as a function of varying Tris or NaI concentrations and at constant 200 mM cosolutes concentration, adjusting NaI and Tris concentrations accordingly. In both cases, we observed strong inhibition of the enzyme with increasing amounts of iodide. The characterization of the κ- and κ-/μ-carrageenan ordering by optical rotation and the visualization of iodide binding on carrageenan by (127)I NMR revealed that inhibition was not caused by the disordered-ordered transition of carrageenan in NaI, but by iodide binding. These results were confirmed by analysis of the degradation products by gel permeation chromatography. Degradation of carrageenan in the disordered state led to a rapid decrease in molecular mass and the production of all possible neo-κ-carrabiose oligomers. In the ordered conformation, the degradation kinetics, the decrease of average molecular weight, and the chain population distribution of degradation products varied with iodide concentration. These observations were interpreted to be the result of increasing amounts of bound iodide on carrageenan helices that, in turn, impede enzyme catalysis. Based on these results, we propose a single-helix ordered conformation state for κ-carrageenan and reject the previously advocated double-helix model.
Collén
Pi Nyvall
PN
Université Pierre et Marie Curie, Paris VI, CNRS, Marine Plants and Biomolecules, UMR 7139, Station Biologique, BP 74, F29680 Roscoff Cedex, France.
Lemoine
Maud
M
Daniellou
Richard
R
Guégan
Jean-Paul
JP
Paoletti
Sergio
S
Helbert
William
W
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002351:Carrageenan / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D031161:Pseudoalteromonas / Q000201:enzymology; D012996:Solutions; D014325:Tromethamine / Q000378:metabolism; D014867:Water
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007454:Iodides; D012996:Solutions; D014325:Tromethamine; D014867:Water; D002351:Carrageenan; D006026:Glycoside Hydrolases; C019846:kappa-carrageenase protein, Alteromonadaceae