Urea-induced denaturation of human phenylalanine hydroxylase

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of th e fusion partner, the effects of increasing urea concentrations on enzyme a ctivity, aggregation, unfolding, and refolding were examined. At pH 7.50, p urified human phenylalanine hydroxylase is transiently activated in the pre sence of 0-4 M urea but slowly inactivated at higher denaturant concentrati ons. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phe nylalanine (L-Phe) shifts the transition midpoint of the first transition f rom 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant d ifferences in d Delta G(D)/d[urea] (m(D) values) were observed for the firs t denaturation transition. In the absence of substrate, a high rate of non- covalent aggregation was observed for the enzyme in the presence of 1-4 M u rea. All three tryptophan residues in the enzyme (Trp-120, Trp 187, and Trp -326) were mutated to phenylalanine, either as single mutations or in combi nation, in order to identify the residues involved in the spectroscopic tra nsitions. A gradual dissociation of the native tetrameric enzyme to increas ingly denatured dimeric and monomeric forms was demonstrated by size exclus ion chromatography in the presence of denaturants.