Site-directed mutants of charged residues in the active site of tyrosine hydroxylase.
The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.
Daubner
S C
SC
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Fitzpatrick
P F
PF
D019943:Amino Acid Substitution / Q000235:genetics*; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D001120:Arginine; D014446:Tyrosine 3-Monooxygenase