PubMed · 6640918
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B Janik. 1983. Gelman kit modified.. https://pubmed.ncbi.nlm.nih.gov/6640918/
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The unfolding of adenylate kinase in urea or guanidine hydrochloride solutions was measured by UV absorbance at 287 nm, circular dichroism at 222 nm and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence. At concentrations less than 1.8 M of urea, the secondary and tertiary structures of AK were not noticeably perturbed. In contrast, the activity of the enzyme underwent significant changes, increasing about 1.6-fold when the urea concentration was increased to 1 M. The enzyme activity then decreased with further increases of the urea concentration. We also observed that the kinetics of ANS binding to AK by fluorescence was biphasic. The fast phase completed within the dead-time of the stopped-flow apparatus used, while the slow phase ended in about 10 minutes. The slow phase fluorescence rate constants increased from 0.0073 s-1 in the absence of denaturants to 0.0100 s-1 (about 1.4-fold) at 1 M urea and then decreased at higher urea concentrations. Similar results were obtained when guanidine hydrochloride was used as a denaturant. The change of the enzyme activity coincided with that of the rate of ANS binding during denaturation by low concentration of denaturants, suggesting that the activation of AK by denaturants may be due to the increasing conformational flexibility at its active site.
The fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS), was used to monitor the induced-fit conformational movement in rabbit muscle adenylate kinase. In 50 mM Tris-HCl buffer (pH 8.1), the time course of ANS binding to rabbit muscle adenylate kinase is a biphasic process. The fast phase completes within the dead-time of the stopped-flow equipment used (about 15 ms), while the slow phase ends in about 10 minutes. In the presence of 2.0 microM peptidyl prolyl cis/trans-isomerase, the rate constant of the slow phase reaction is accelerated about 2.4-fold, suggesting that the domain movement during ANS binding to rabbit muscle adenylate kinase may involve proline isomerization. The activation energy of the slow phase was determined to be 74.6 kJ/mol, which is comparable to the activation energy of proline cis/trans-isomerization (about 80 kJ/mol).
Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.