Active site structure determination spin-labeling

To this end, the spin labeling technique was used to probe the active site structure of LADH in various solvent systems. The spin label, SL-1, alkylated cysteine 46 (12), an amino acid in the active site of LADH that normally serves as a ligand to the catalytic zinc ion. LADH is a dimer, and each monomeric subunit contains one firmly-bound catalytic zinc ion. The position of enzyme-bound SL-1 was estimated by the spin label-spin probe technique (14.15). Cobalt (II) was employed as the spin probe, and the catalytic zinc in the enzyme s active site was replaced by Co + according to the procedure of Sytkowski and Vallee (16). The EPR spectrum of enzyme-bound SL-1 was then measured before and after replacement of the active-site zinc by cobalt from the decrease in spectral amplitude the average nitroxide-metal distance was determined to be 4.8 1.5 A (17). 

The coordination structures of enzyme-bound manganese nucleotides can in favorable cases be determined by analysis of electron paramagnetic resonance (EPR) spectra of Mn(II) coordinated to O-labeled nucleotides. When the nucleotide is stereospecifically labeled with O at one diastereotopic position of a prochiral center, either oxygen can in principle be bound to Mn(II) in the coordination complex in an enzymic site. When the coordination bond is between Mn(II) and O, the EPR signals for Mn(II) are broadened and attenuated, owing to unresolved superhyperfine coupling between the nucleus of 0 and the unpaired electrons of Mn(II) (23). No such effect is possible with 0, which has no nuclear spin. The effect is observable in samples in which all the Mn(Il) is specifically bound in one or two defined complexes of the nucleotide with the enzyme. Thus the complex Mg(Sp)-[a- 0]ADP bound at the active site of ere-... 

Distances between the nitroxide of bound steroid and several assigned protons in or near the active site could be determined on the basis of the paramagnetic effects of the nitroxide on the relaxation rates of the active site protons. These distances could then be used to dock the structure of the spin-labeled steroid into a partially refined 2.5-A resolution X-ray structure of the isomerase 128). However, in order to rationalize the chemical evidence that Asp-38 is involved in the catalytic mechanism, it was necessary to assume that steroid substrates and the spin-labeled steroid (16) are bound not only with reversal of the C-3 and C-17 positions, but also with reversal of the planes of the steroid ring systems ( up-sidedown binding), positioning Asp-38 above the C-4j8 and C-6j3 protons. 

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