Thermodynamic, Kinetic and pH Studies on the Reactions of NCS^-, N₃^-, and CH₃CO₂^- with Fusarium Galactose Oxidase

Thermodynamic and kinetic studies on the X^- = NCS^-, N₃^-, and CH₃CO₂^- replacement of H₂O/OH^- at the Cu^II exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOase_ox) from Fusarium (NRR 2903), have been studied by methods involving UV−vis spectrophotometry (25 °C), pH range 5.5−8.7, I = 0.100 M (NaCl). In the case of N₃^- and CH₃CO₂^- previous X-ray structures have confirmed coordination at the exogenous H₂O/OH^- site. From the effect of pH on the UV−vis spectrum of GOase_ox under buffer-free conditions, acid dissociation constants of 5.7 (pK_1a; coordinated H₂O) and 7.0 (pK_2a; H⁺Tyr-495) have been determined. At pH 7.0 formation constants K(25 °C)/M^-1 are NCS^- (480), N₃^- (1.98 × 10⁴), and CH₃CO₂^- (104), and from the variations in K with pH the same two pK_a values are seen to apply. No pK_1a is observed when X^- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction k_f(25 °C)/M^-1 s^-1 are NCS^- (1.13 × 10⁴) and N₃^- (5.2 × 10⁵). Both K and k_f decrease with increasing pH, consistent with the electrostatic effect of replacing H₂O by OH^-. In the case of the GOase_ox Tyr495Phe variant pK_1a is again 5.7, but no pK_2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOase_ox Tyr495Phe with NCS^- (1.02 × 10⁵M^-1) is more favorable than the value for GOase_ox. Effects of H⁺Tyr-495 deprotonation on K are smaller than those for the H₂O/OH^- change. The pK_1a for GOase_semi is very similar (5.6) to that for GOase_ox (both at Cu^II), but pK_2a is 8.0. At pH 7.0 values of K for GOase_semi are NCS^- (270 M^-1), N₃^- (4.9 × 10³), and CH₃CO₂^- (107).