文摘
The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactoseoxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPRspectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnoveris significantly slower in the W290 variants. The Km for D-galactose for W290H is similar to that of thewild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 insubstrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding betweenW290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulatesthe properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both theW290G and W290H variants have elevated redox potentials and are highly unstable compared to theradical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restrictsthe accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantlymore solvent exposed in the W290G variant but that W290F limits solvent access comparable to thewild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireducedW290 variants are very similar to that of the wild-type protein. In addition, the electronic structures ofW290X-azide complexes are also closely similar to the wild-type electronic structure. Azide bindingand azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan alsomodulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiplecritical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability,and the axial tyrosine function.