Forms Of Adenylate Kinase Research Articles

Multiple Pathways and Time Scales for Conformational Transitions in apo-Adenylate Kinase.

The open/close transition in adenylate kinase (AK) is regarded as a representative example for large-scale conformational transition in proteins, yet its mechanism remains unclear despite numerous experimental and computational studies. Using extensive (∼50 μs) explicit solvent atomistic simulations and Markov state analysis, we shed new lights on the mechanism of this transition in the apo form of AK. The closed basin of apo AK features an open NMP domain while the LID domain closes and rotates toward it. Therefore, although the computed structural properties of the closed ensemble are consistent with previously reported FRET and PRE measurements, our simulations suggest that NMP closure is likely to follow AMP binding, in contrast to the previous interpretation of FRET and PRE data that the apo state was able to sample the fully closed conformation for "ligand selection". The closed state ensemble is found to be kinetically heterogeneous; multiple pathways and time scales are associated with the open/close transition, providing new clues to the disparate time scales observed in different experiments. Besides interdomain interactions, a novel mutual information analysis identifies specific intradomain interactions that correlate strongly to transition kinetics, supporting observations from previous chimera experiments. While our results underscore the role of internal domain properties in determining the kinetics of open/close transition in apo AK, no evidence is observed for any significant degree of local unfolding during the transition. These observations about AK have general implications to our view of conformational states, transition pathways, and time scales of conformational changes in proteins. The key features and time scales of observed transition pathways are robust and similar from simulations using two popular fixed charge force fields.

Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibriogigas

Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co 2+/Zn 2+-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co 2+-, Zn 2+- and Fe 2+-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co 2+- and Zn 2+-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe 2+-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe 2+-AK, in contrast to Co 2+- and Zn 2+-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.

Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis.

Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.

Native states of adenylate kinase are two active sub-ensembles

There are two kinds of conformational forms of adenylate kinase (AK) in equilibrium in solution with different ANS-binding properties. Furthermore, the nature of AP 5A inhibition suggests also that the native forms of AK for binding with different substrates pre-exist in the absence of substrates. In the present study, a kinetics approach was used to explore the native forms distinguished by ANS-binding properties and by the nature of AP 5A inhibition. The results revealed that the native forms distinguished by ANS probe are two conformational sub-ensembles. Both sub-ensembles are active and consist of a series of forms, which pre-exist in solution and can bind with different substrates. The K m values of N 1 for AMP, ADP and MgATP are larger than that of N 2, indicating that the N 2 sub-ensemble is more specific for binding substrates. This is consistent with the previous observation that the activity of N 2 is about 1.8-fold of that of N 1.

Both native conformers of rabbit muscle adenylate kinase are active

There are two forms of rabbit muscle adenylate kinase (AK) with different 8-anilino-1-naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N 1) binds rapidly with ANS, whereas the other (about 30%, denoted N 2) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre-existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N 2 is about 0.8 fold higher than N 1 and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum.

Tissue determined variations of adenylate kinase.

GENETICALLY determined isoenzymes of human erythrocyte adenylate kinase (AK), ATP : AMP phosphotransferase E.C. 2.7.4.3, have been reported1,2. We have characterized several forms of AK in different organs of Wistar rats, employing a mixed agarose-acrylamide gel electrophoresis3. The electrophoretic behaviour of AK was studied in tissues taken from the same animal. Extracts of muscle, heart, eye lens, brain, liver and erythrocytes have been compared.

Adenosine Triphosphate-Adenosine 5'-Monophosphate Phosphotransferase of Bovine Liver Mitochondria: II. GENERAL KINETIC AND STRUCTURAL PROPERTIES

Some of the kinetic and structural properties of purified adenylate kinase derived from bovine liver mitochondria have been investigated and compared with the corresponding properties of adenylate kinase from rabbit muscle. The results of studies of the isotope exchange reaction between each of the substrates at equilibrium are consistent with the two-site mechanism proposed for the skeletal muscle enzyme. Other studies show that the liver enzyme is similar to the muscle enzyme with respect to maximum catalytic rate, Km values of the several substrates, product inhibition, cation activation, and pH optimum, and therefore provide evidence that both enzymes utilize a similar reaction mechanism. Studies of the structural properties of the liver enzyme have confirmed earlier suggestions that it does not contain an essential sulfhydryl group but does contain at least one and probably two disulfide bridges which are essential for enzymatic activity. The presence of essential disulfide groups was inferred from the loss of activity when the enzyme was treated with reagents such as sodium sulfite or β-mercaptoethanol. The inactivation observed with β-mercaptoethanol could be reversed by exposure to air, which presumably results in reoxidation of the reduced disulfide bridge, or bridges. The results described in this and the preceding communication indicate that different, possibly tissue-specific, forms of adenylate kinase do exist in mammalian tissues and, therefore, that this enzyme constitutes an addition to a growing list of enzymes that exist in multiple molecular forms.