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| - Effects of Ligands on the Mobility of an Active-Site Loop in Tyrosine Hydroxylaseas Monitored by Fluorescence Anisotropy
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| - Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop overthe active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, andthe three native tryptophan residues were mutated to phenylalanine to form an enzyme with a singletryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzymeresulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did notresult in a significant change in the anisotropy in the presence or absence of the deazapterin. The Kdvalue for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar resultswere obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease inanisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes wasunaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistentwith a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could bemodeled as a decrease in the cone angle for the indole ring of about 12°. The data are consistent with amodel in which binding of a tetrahydropterin results in a change in the conformation of the surface looprequired for proper formation of the amino acid binding site.
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