Abstract
The pH optimum of rat liver phenylalanine hydroxylase is dependent on the structure of the cofactor employed and on the state of activation of the enzyme. The tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase has a pH optimum of about 8.5. In contrast, the 6,7-dimethyltetrahydropterin-dependent activity is highest at pH 7.0. Activation of phenylalanine hydroxylase either by preincubation with phenylalanine or by limited proteolysis results in a shift of the pH optimum of the tetrahydrobiopterin-dependent activity to pH 7.0. Activation of the enzyme has no effect on the optimal pH of the 6,7-dimethyltetrahydropterin-dependent activity. The different pH optimum of the tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase is due to a change in the properties of the enzyme when the pH is increased from pH 7 to 9.5. Phenylalanine hydroxylase at alkaline pH appears to be in an altered conformation that is very similar to that of the enzyme which has been activated by preincubation with phenylalanine as determined by changes in the intrinsic protein fluorescence spectrum of the enzyme. Furthermore, phenylalanine hydroxylase which has been preincubated at an alkaline pH in the absence of phenylalanine and subsequently assayed at pH 7.0 in the presence of phenylalanine shows an increase in tetrahydrobiopterin-dependent activity similar to that exhibited by the enzyme which has been activated by preincubation with phenylalanine at neutral pH. Activation of the enzyme also occurs when m-tyrosine or tryptophan replace phenylalanine in the assay mixture. The predominant cause of the increase in activity of the enzyme immediately following preincubation at alkaline pH appears to be the increase in the rate of activation by the amino acid substrate. However, in the absence of substrate activation, phenylalanine hydroxylase preincubated at alkaline pH displays an approximately 2-fold greater intrinsic activity than the native enzyme.
Highlights
From the Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20205
Phenylalanine hydroxylase at alkaline pH appears proteolysis (4, 17), andsulfhydryl modification (18), resultin to be in an altered conformation tihsavtery similar to the abolition of the positive homotropic efthat of the enzyme which hasbeen activated by prein- fect of phenylalanine
The variation of VmaXas a function of pH was found to be different when native phenylalanine hydroxylase2was assayed with BH, or with DMPH
Summary
The variation of VmaXas a function of pH was found to be different when native phenylalanine hydroxylase2was assayed with BH, or with DMPH,. It has been reported that there is a greater than 10-fold increase in BH,-dependent activity when the enzyme is assayed at pH 6.8, following a 10-min preincubation with 1mM phenylalanine prior to the addition of the pterin cofactor (9, 12).An identical preincubation had no effect on the DMPH,dependent activity of the hydroxylase (data notshown). When assayed at pH 6.8, the activity of phenylalanine hydroxylase that had been incubated at pH 9.3 (Fig. 3, middle curve) is between that of the control enzyme and thephenylalanine-activated enzyme This activation due to exposure to pH9.3 is transient asseen by the upward curvature of the reaction trace at relatively early times after mixing. The emission spectrum of phenylalanine hydroxylase at pH 9.5 changes only very slightly upon the addition of 1.2 mM phenylalanine (Fig. 4B) These resultsindicate that preincubation of phenylalanine hydroxylase at alkaline pH activates the enzyme in an analogous manner to thasteen during activation by phenylalanine.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
