title: Structural changes of rabbit liver fructose-1,6-bisphosphatase: the effect of urea and subtilisin digestion.
abstract: At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.
authors.forename: A
authors.initials: A
authors.lastname: Dzugaj
authors.forename: T
authors.initials: T
authors.lastname: Heyduk
authors.forename: J
authors.initials: J
authors.lastname: Buczyłko
authors.forename: M
authors.initials: M
authors.lastname: Kochman
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism*; D013997:Time Factors; D014508:Urea / Q000494:pharmacology*
chemical_list: D000249:Adenosine Monophosphate; D014508:Urea; D006597:Fructose-Bisphosphatase; D013381:Subtilisins