The Metal-Binding Site

Galactose oxidase hinds a single copper ion within Domain 11 on the axis of the wheel. The active site (Fig. 5) is unhke any other biological copper complex, an appropriate distinction for this remarkable enzyme. To explore the site in more detail, the protein environment of the mononuclear copper center may be separated into (A) direcdy coordinated metal hgands (hrst shell, inner sphere interactions) and (B) the extended active site environment (the second shell or outer coordination sphere). 

Trp-290 affects the stability of the active enzyme and ligand interactions (Reynolds et al., 1997). 

For GAOX, structural analysis is particularly complicated, because of the existence of multiple states of the enzyme differing essentially only in the number of electrons, i.e., the oxidation state of the metalloprotein complex. Three distinct oxidation states can be prepared, each with properties and reactivities dramatically different from the others, as indicated in Fig. 10 (Whittaker and Whittaker, 1988). When isolated from culture medium, GAOX is a mixture of two of these states a blue, one-electron reduced, catalytically inactive form (lAGO) that contains a Cu(II) ion and no radical and a green form that is catalytically active (AGO) and contains both Cu(II) and a free radical. The enzyme may be converted to 

Optical spectroscopy involves electronic excitations in molecules, making it well suited to extending the atomic structural information 

The active enzyme (AGO) is distinguished by an unusual spectrum (Fig. 11, line A) unlike spectra observed for either RGO or lAGO forms, with extremely strong absorption that spans the entire visible region and extends deep into the near infrared. The principal absorption in this form is associated with two intense features centered at 445 and 850 nm. This spectrum is, in fact, unlike spectra observed for any other metaUoprotein complex, emphasizing the unique character of the GAOX active site. The origin of this spectrum is discussed in more detail below (Section VII). 

---

The dependence of chiral recognition on the formation of the diastereomeric complex imposes constraints on the proximity of the metal binding sites, usually either an hydroxy or an amine a to a carboxyHc acid, in the analyte. Principal advantages of this technique include the abiHty to assign configuration in the absence of standards, enantioresolve non aromatic analytes, use aqueous mobile phases, acquire a stationary phase with the opposite enantioselectivity, and predict the likelihood of successful chiral resolution for a given analyte based on a weU-understood chiral recognition mechanism. 

In the active site of PDF proteins, three substrate-binding pockets exist along with the metal-binding site. Using standard metalloprotease nomenclature. 

Some microbes are able to decrease the permeability of their membranes to prevent toxic metals from entering. If the toxic metals are not able to physically enter the cell, they will not be able to affect vital metal-sensitive structures, such as proteins. One way to prevent heavy metals from entering is by decreasing the production of membrane channel proteins.18 It is also possible for the metal-binding sites in the membrane and periplasm to be saturated with nontoxic metals.37 A third possibility is the formation of an extracellular polysaccharide coat, which binds and prevents metals from reaching the surface of the cell.24,38... 

ESE envelope modulation studies of a number of Cu(II) compounds have been reviewed by Peisach1171. The aim of these investigations was to characterize the chemical environment of the metal binding site in Cu(II) proteins by comparison of the nuclear modulation pattern with those for Cu(II) complexes of known composition. 

Vance and Miller et al. have shown that the inactivity of enzyme is due to changes in the redox potentials of the enzyme. In order to dismute 02 the redox potential of the enzyme must lie between the E° values for the reactions shown in Equation (3). The E° value of the E. coli MnSOD enzyme is 0.290 V and that for the FeSOD is 0.220 V. The Fe-substituted form of the Mn enzyme has F ° =—0.240 V and Mn-substituted FeSOD has ii° >0.960V. These values are outside the required range and the changes in redox potentials are not due to changes in the metal ligands. Mutations of His-30 and Tyr-34, two conserved residues in the immediate vicinity of the metal binding site, do not alter the redox potential of the enzyme either " ... 

Figure 29 Schematic view of the metal binding site of MECDP synthase.

Arginine is capable of binding in the active site of PAH but it is not an enzyme substrate. Crystal structures of PAH from Streptomyces clavuligerus show that the metal binding site is very similar to that of arginase and the Mn—Mn distance is 3.3 A The main difference between arginase and PAH is in the binding pocket at the o-amino-terminus of the substrate. ... 

In the metal-binding site (Figure 12) the Cu(II) ion is coordinated by four histidines in a distorted square-planar coordination sphere and by a fifth axial water ligand, while the tetrahedral zinc ion is coordinated by three histidines, one of which is deprotonated and bridges to the copper, and by a carboxylate group °. 

---