Role of tryptophan 161 in catalysis by human manganese superoxide dismutase.
Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 A from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O(2)(*)(-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 A from the manganese, closer by 1.2 A than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese; the mutant W161F MnSOD had k(cat)/K(m) at 2.5 x 10(8) M(-)(1) s(-)(1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O(2)(*)(-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O(2)(*)(-) with the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34.
Cabelli
D E
DE
Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973-5000, USA. cabelli@bnl.gov
Guan
Y
Y
Leveque
V
V
Hearn
A S
AS
Tainer
J A
JA
Nick
H S
HS
Silverman
D N
DN
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D014364:Tryptophan; D013482:Superoxide Dismutase