Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus.
The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.
Holzwarth
J F
JF
Fritz-Haber-Institut, Max-Planck-Gesellschaft, Berlin, West Germany.
Meyer
F
F
Pickard
M
M
Dunford
H B
HB
D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002711:Chloride Peroxidase / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007455:Iodine; D007700:Kinetics; D007834:Lasers; D008433:Mathematics; D003904:Mitosporic Fungi / Q000201:enzymology; D008962:Models, Theoretical; D010544:Peroxidases / Q000378:metabolism*; D011485:Protein Binding; D019810:Sodium Azide; D013816:Thermodynamics
D001386:Azides; D007455:Iodine; D019810:Sodium Azide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002711:Chloride Peroxidase