Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Abstract

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 A from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity ( k(cat)) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K(M) values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3-4 in the Y179F mutant. However, the K(M) values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K(M) value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k(cat)/ K(Phe), the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.

Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

“…4(a). This observation agrees with previous data on the parameters of activation for cPAH [18]. The K M of the enzyme for Phe did not vary significantly between 20 and 40°C as shown in Fig.…”

The active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylase

“…The most deleterious consequences of mutating Tyr325 are in fact the loss of iron and the uncoupling of the reaction. Loss of iron has been shown to result in a decreased conformational stability of the protein both for bacterial (C. violaceum) PAH [11] and for tyrosine hydroxylase [33]. The effects of the mutation are thus compatible with the important structural role of this residue by determining the close structure of the active site around the metal ( Fig.…”

“…The posttranslational oxidative repair of the Y325F PAH mutant to retain wild-type structure again indicates the functional importance of Tyr325. Accordingly, the mutation of Tyr to Phe in the corresponding residue (Tyr179) in Chromobacterium violaceum PAH, which is not the subject of self hydroxylation, significantly decreases the catalytic activity while increasing the apparent affinity for L-Phe [11]. The same catalytic effects accompanied the mutation of Y325L in rat PAH [7].…”

Rational design, synthesis and characterization of amide functionalized pyridine and benzimidazole transition metal complexes.

“…12450 (40) 4157 (7) 8836 (3) 27(3) C (12) 10580 ( (19) 9130 (30) 5949 (7) 6745 (3) 17(1) C (20) 9520 (30) 6272 (7) 6448 (3) 17(1) C (21) 7910 (30) 6882 (6) 6348 (3) 17(1) C (22) 5800 (30) 7164 (7) 6540 (3) 17(1) C (23) 5440 (30) 6834 (7) 6839 (3) 17(1) C (24) 4070 (30) 6509 (7) 7336 (3) 17(1) C (25) 3590 (20) 6244 (6) 7883 (3) 14(2) C (26) 2010 (20) 6358 (6) 8193 (3) 14(2) C (27) 3750 (30) 6328 (7) 8495(3) 20(3) C (28) 2060 (30) 6377 (7) 8805 (3) 18(3) C (29) 3810 (30) 6332 (8) 9109 (3) 23(3) C (30) 2150 (40) 6415 (7) 9422(3) 24(3) C (31) 3930 …”

“…Superoxide dismutase catalyzes the conversion of superoxide into hydrogen peroxide and oxygen [14,[18][19][20][21][22][23][24][25]. Without the action of superoxide dismutase and a cascade of enzymes, the build up of superoxide, generated during the production of ATP, leads to oxidative stress.…”