Chemical modification was used to examine the role of some amino acid residues in the binding of the substrates to the enzyme p-hydroxybenzoate hydroxylase from <em>Pseudomonas fluorescens.</em> Ionic strength dependent binding studies were used to investigate the role of the protein as a whole in the complex formations. In the general introduction the present knowledge on p-hydroxybenzoate hydroxylase and on chemical modifications of amino acid residues in general is reviewed. Whereas most studies on this enzyme were done on the prosthetic group FAD, it was our aim to acquire more insight into the role of the surrounding protein.<p/>Diethylpyrocarbonate was used to modify histidine residues. Four out of nine histidine residues were modified, which was accompanied by total inactivation of the enzyme. Two of these residues appeared to be essential for the enzyme activity and to be involved in the binding of NADPH. The rate of inactivation increased with increasing pH. From this pH dependence the pK value of two cooperatively ionizing histidine <em>residues was</em> determined. At pH>7 tyrosine residues appeared to be carbethoxylated, accompanied by a loss of substrate binding capacity.<p/>The modification of arginine residues is described in chapter 3. Out of the three inactivating reagents used, one appeared to react with two arginine residues which are important for NADPH and substrate binding. A model was postulated to account for the results. The other two reagents reacted with different arginine residues, which was explained by the differences in charge and hydrophobicity. The complexity of arginine modification is due to the large number (38) of arginine residues present in the enzyme.<p/>In chapter 4 the modification of tyrosine residues is described. Some additional experiments using diethylpyrocarbonate were carried out, but more attention is given to the modification by the substrate analogue p-diazobenzoate. Although the identification experiments did not yield the desired quantitative result, it could be established that a reaction with tyrosine-201 was responsible for the loss of substrate binding capacity. It was concluded that tyrosine-385 is also essential and that tyrosine-222 is non-essential.<p/>In chapter 5 the protein as a whole is considered. The ionic strength dependence of NADPH binding to the enzyme is explained using a model in which the enzyme is considered as both a monopole and a dipole. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated and compared with a dipole moment estimated on the basis of charge distribution as revealed by the three-dimensional model. The apparent contradiction between the pH dependences of NADPH binding and enzyme activity are also discussed.<p/>Chapter 6 finally is dedicated to the results of time resolved flavin fluorescence measurements on enzyme molecules modified by various reagents. The rotation correlation times determined were compared to obtain information on the mobility of the flavin within the enzyme. The differences between the rotation correlation times could be accounted for by a shift of the dissociation constant of the dimeric form of p-hydroxybenzoate hydroxylase.
|Qualification||Doctor of Philosophy|
|Award date||28 Nov 1984|
|Place of Publication||Wageningen|
|Publication status||Published - 1984|
- enzyme activity