Rheo-NMR studies of an enzymatic reaction: evidence of a shear-stable macromolecular system.
Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, (1)H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s(-1) did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely beta-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.
Edwards
Patrick J B
PJ
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Kakubayashi
Motoko
M
Dykstra
Robin
R
Pascal
Steven M
SM
Williams
Martin A K
MA
D001234:Aspergillus niger / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D010368:Pectins / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D011487:Protein Conformation
D004798:Enzymes; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase