Spectral studies of conformational changes induced in beta-glucuronidase by saccharo-1,4-lactone.
Ultraviolet difference spectroscopy has been used to study the binding of the transition state analog saccharo-1,4-lactone to purified rat preputial gland beta-glucuronidase. At pH 4.5 (the pH optimum), the inhibitor induces a difference spectrum indicative of a change in the environment of tryptophyl residues. Based on the magnitude of the induced difference spectrum as a quantitative measure of inhibitor binding, a titration curve for saccharo-1,4-lactone was obtained. A Scatchard plot of the titration data indicates that 4 molecules of inhibitor bind to the enzyme tetramer at a K-I of 4 times 10-7 M. The inhibitor also induces a similar difference spectrum at pH 7.5, although the binding is considerably weaker at this pH than at pH 4.5. When the native enzyme at pH 4.5 is compared with the native enzyme at pH 7.5, a difference spectrum, distinct from that of the binding of saccharo-1,4-lactone, is observed, indicating that the enzyme exists in different conformations at these pH values. The indication that tryptophyl residues are perturbed upon binding of saccharo-1,4-lactone was supported by studies carried out with N-bromosuccinimide. At pH 4.3, this reagent was found to oxidize 6 tryptophyl residues in the native enzyme but only three in the saccharo-1,4-lactone-inhibited enzyme. A spectrophotometric titration of the enzyme indicated that of the 33 tyrosyl residues per subunit, only 5 to 6 ionize at the pK expected for free phenolic groups.
D000272:Adipates; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D005690:Galactose; D005966:Glucuronidase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012627:Sebaceous Glands / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides; D013400:Sugar Acids; D014364:Tryptophan / Q000032:analysis
D000272:Adipates; D007783:Lactones; D013388:Succinimides; D013400:Sugar Acids; D014364:Tryptophan; D005966:Glucuronidase; D005690:Galactose