Paramagnetic probes, of enzyme complexes

Paramagnetic Probes of Enzyme Complexes with Phosphorus-Containing Compounds... 

The present paper will outline some of our recent EPR and NMR studies using Mn2+ as a paramagnetic probe of sheep kidney (Na+ + K+)-ATPase and Gd2+ as a paramagnetic probe of sarcoplasmic reticulum Ca2+-ATPase. Estimates of the relevant electron spin relaxation times and some features of the interaction between substrates and activators with the enzyme-metal complexes will be inferred from the EPR spectra and the accompanying nuclear relaxation data. 

Exchange-inert complexes of Cr(III) with nucleotide ligands are very stable toward hydrolysis. Such complexes have proven to be extremely useful as chirality probes in that different coordination isomers can be prepared and separated These nucleotide complexes have also proved useful as dead-end inhibitors of enzyme-catalyzed reactions. Because Cr(lII) is paramagnetic, distances can be measured by measuring the effects of Cr(lll) on the NMR signals of nearby atoms when the Cr(lll)-nucleotide complex binds to the surface of a mac-romolecule. See Exchange-Inert Complexes... 

Nucleotides in the form of metal ion complexes are involved in a variety of enzymatic reactions either as substrates or as cofactors. These may also be viewed as monomers of DNA and RNA. Lanthanide complexes of nucleotides have been extensively studied because (i) the conformation of nucleotides in solution can be elucidated from lanthanide induced NMR chemical shifts and line-broadenings [40] and (ii) lanthanide nucleotide complexes may act as competitive inhibitors in enzymatic reactions [88] and hence can be used as paramagnetic probes in the mapping of their binding site on the enzyme [89]. 

As yet, no X-ray crystal structures are available for any of the molybdenum enzymes in Table I. Therefore, present descriptions of the coordination environment of the molybdenum centers of the enzymes rest primarily upon comparisons of the spectra of the enzymes with the spectra of well-characterized molybdenum complexes. The two most powerful techniques for directly probing the molybdenum centers of enzymes are electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS), especially the extended X-ray absorption fine structure (EXAFS) from experiments at the Mo K-absorption edge. Brief summaries of techniques are presented in this section, followed by specific results for sulfite oxidase (Section III.B), xanthine oxidase (Section III.C), and model compounds (Section IV). 

CrATP has been used with several enzymes as a paramagnetic probe (Gupta et al, 1976 Balakrishnan and Villafianca, 1978a Villafianca, 1980). For some cases a mixture of isomers was used for distance determinations, and one isomer could have bound preferentially to the enzyme that would lead to an error in determining/ the mole ficaction of paramagnetic species in the enzyme complex. Another problem is that the t, value for Cr is 2 X 10 ° s. Thus the paramagnetic influence of Cr on the H or other nuclei of ligands is quite often small. Distances > 8 A from Cr + to a nucleus are not well determined and most often must be viewed with caution. 

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