Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase.
The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.
D002730:Chloromercurinitrophenols; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D010735:Phosphoglycerate Kinase; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013057:Spectrum Analysis; D014508:Urea / Q000494:pharmacology*
D002730:Chloromercurinitrophenols; D014508:Urea; D010735:Phosphoglycerate Kinase