Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.
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ABSTRACT:
SUBMITTER: Bray RC
PROVIDER: S-EPMC1184010 | biostudies-other | 1978 Jun
REPOSITORIES: biostudies-other
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Project description:Further electron-paramagnetic-resonance studies relating to the role of molybdenum in the enzymic mechanisms of xanthine oxidase were carried out. The classification of the various molybdenum signals obtained on reducing the enzyme is briefly discussed. The group of ;Rapidly appearing' signals, which are obtained with all substrates within the turnover time and which show interaction with exchangeable protons, were studied in detail. Signals with salicylaldehyde, purine and xanthine in H(2)O and in 95% D(2)O were examined at 9 and 35GHz and interpreted with the help of computer simulation. Molybdenum atoms in a number of different chemical environments are involved, each substrate giving rise to two superimposed spectra with slightly different parameters; g values and proton splittings were determined. The spectrum with salicylaldehyde is believed to represent the reduced enzyme alone not in the form of a complex with substrate and its two constituents are believed to represent the two molybdenum atoms bonded slightly differently within the enzyme molecule. With purine and xanthine the spectra are thought to represent complexes of reduced enzyme with substrate molecules. With xanthine one signal-giving species shows coupling to two equivalent protons, whereas in all the other species observed two non-equivalent protons are involved. The origin of the protons is discussed in the light of the direct hydrogen-transfer mechanism implicated earlier for the enzyme. It is concluded that the proton derived from the substrate is located at least 3å from the molybdenum atom with which it interacts.
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Project description:Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca(2+))(4-)form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2-3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca(2+) to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca(2+) binding. The midpoint of the Ca(2+)-mediated transition determined by EPR occurred at a higher Ca(2+) concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca(2+))(4-)CaM], contains a compact intermediate corresponding to [(Ca(2+))(2-)CaM], and the present results suggest that the spin probes are reporting on Ca(2+) binding to the last two sites in the N-terminal domain, i.e. for the [(Ca(2+))(2)-CaM] ? [(Ca(2+))(4-)CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca(2+)-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.
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