Acid-Induced Folding of Yeast Alcohol Dehydrogenase under Low pH Conditions

Abstract

Under conditions of low pH, the conformational states of holo-YADH and apo-YADH were examined by protein intrinsic fluorescence, ANS fluorescence, and far-UV CD measurements. The results obtained show that a low ionic strength, with the addition of HCl, the holo- and apo- YADH denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 2.0 and 2.5, respectively. With the decrease of pH from 7.0 to 2.0, the fluorescence emission decreased markedly, with its emission maximum red-shifting from 335 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far-UV CD spectra show the loss of the arrayed secondary structure, though the acid-denatured enzyme still maintained a partially arrayed secondary structure. A further decrease in pH by increasing the concentration of HClO4 induced a cooperative folding of the denatured enzyme to a compact conformation with the properties of a molten globule, described previously by Goto et al. [Proc. Natl. Acad. Sci. USA 87, 573-577 (1990)]. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-YADH was lower than that of the holo-enzyme. From the above results, it is suggested that the zinc ion plays an important role in the proper folding of YADH and in stabilizing its native conformation.

Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time‐of‐flight mass spectrometry

“…Interestingly, apo‐E9 DNase at low pH had negative ellipticity at 222 nm, whereas the near‐UV CD signal and the Trp fluorescence were lost. Thus, at least under acidic conditions, apo‐E9 DNase seems to adopt features that are reminiscent of molten globule‐like states, that is, states that lack a fixed tertiary structure but in which secondary structure is retained (Le et al 1996; Chak et al 1998). In this regard, it is intriguing to speculate that the local acidic pH at the membrane surface triggers metal ion release and concomitantly renders the DNase in a molten globule‐like state competent for membrane translocation (Prats et al 1986; McLaughlin 1989; Bychkova et al 1996).…”

Unassisted refolding of urea‐denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

“…A commonly observed equilibrium intermediate, termed the “molten globule” state (MG), is characterized by a pronounced secondary structure, a compact globular shape, exposure of the hydrophobic core to solvent, and the absence of rigid side‐chain packing (Ptitsyn 1987, 1995; Kuwajima 1989, 1996). MGs are frequently detected in the process of denaturation and refolding of protein (Le et al 1996; Bai et al 1999).…”

Yeast‐mediated enantioselective synthesis of chiral R‐(+)‐ and S‐(−)‐1‐phenyl‐1‐butanol from prochiral phenyl n‐propyl ketone in hexane–water biphasic culture

“…The pH values tested for the reaction were 4.0, 5.0, 6.0, 7.0 and 8.0 with a culture of 0% hexane volume percentage, which avoids poisoning of the cell and denaturation of alcohol dehydrogenase and reduces the cost of the experiment. Because Zn 2+ ion is an important cofactor in stabilizing the native conformation of alcohol dehydrogenase,27, 28 we arbitrarily added 0.1 mg ZnSO 4 to the reaction culture for these experiments. Table 2 gives the % e.e.…”

“…3(a). Therefore, the presence of Zn 2+ ion could be important in three ways: (1) it can bind the substrate in a specific spatial position; (2) it can extract the substrate from the hexane layer; and (3) it can stabilize the enzyme conformation 27, 28, 32…”