Acid pH-induced conformational changes in bovine liver rhodanese.
The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.
Horowitz
P M
PM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Xu
R
R
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D009038:Motion; D010434:Pepsin A / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D013884:Thiosulfate Sulfurtransferase; D010434:Pepsin A