|
Ren et al. 10.1073/pnas.0407459102. |
Fig. 4. Sequence analysis defines a family of adenylate kinases. Shown are structure-based multiple sequence alignments of eukaryotic adenylate kinase 6 (AK6) homologs. The sequences, searched by using European Molecular Biology Laboratory-European Biotechnology Institute FASTA (www.ebi.ac.uk/fasta33), are human (AAF14860), rabbit (AAF09498), mouse (NP_081868), rat (XP_215465), rice (BAC84255), Arabidopsis (BAC42255), Drosophila (NP_610797), Caenorhabditis elegans (NP_496065), Fission yeast (T41637), and Baker’s yeast (NP_010115). The percentage of sequence identities between the human and the other AK6 are shown in brackets after the organism names. Some AK6 sequences, such as rice, Arabidopsis, C. elegans, and Baker’s yeast (Fap7p), contain a nuclear location sequence (NLS) at the N terminus.
Fig. 5. Autoradiogram showing substrates phosphorylated by adenylate kinase 6. The nucleoside monophosphates are AMP (lane 1), dAMP (lane 2), CMP (lane 3), dCMP (lane 4), GMP (lane 5), dGMP (lane 6), IMP (lane 7), dIMP (lane 8), UMP (lane 9), TMP (lane 10), negative control (lane 11). The nucleoside diphosphate products have identical Rf values as the nonradioactive standards.
Table 2. Phosphate donor specificity of adenylate kinase 6 (the AD-004 protein)
|
Phosphate donor |
Activity, nmol∙min–1·mg–1 |
|
ATP |
158 ± 4.0 (100) |
|
UTP |
524 ± 14 (331) |
|
GTP |
153 ± 10 (97) |
|
CTP |
828 ± 40 (523) |
|
dATP |
35 ± 2.0 (22) |
|
dTTP |
23 ± 2.5 (14) |
|
dCTP |
211 ± 9.2 (133) |
|
dGTP |
12 ± 0.4 (35) |
Assays were performed with 100 m M [3H]AMP and 5 mM of each nucleoside triphosphate. The data were the mean values of three independent measurements. Numbers in parentheses are the percentages of the activity with ATP (set to 100%) as phosphate donor.
Table 3. Kinetic parameters for adenylate kinase 6 with the substrates AMP and ATP
|
Variable substrate |
Fixed substrate |
Km, m M |
Vmax, nmol∙min–1·mg–1 |
|
AMP (5-1005 m M) |
ATP (2 mM) |
192 ± 5 |
955 ± 24 |
|
ATP (50-2000 m M) |
AMP (1 mM) |
44 ± 2 |
982 ± 30 |
The Km and Vmax values were calculated using the Michaelis–Menten equation, and the results were from at least three independent measurements.
Fig. 6. Structural comparison of human adenylate kinase (AK)6 with other AKs. (A) Catraces of superposed human AK6 with AK1 (PDB ID code 3ADK, porcine), AK2 (PDB ID code 2AK2, bovine) and AK3 (PDB ID code 2AK3, bovine). Caatoms of the five parallel stands in the core regions were used for the rms deviation calculations. (B) The superposition of the active sites of the human AK6 with that of an "open form" of porcine AK1 (PDB ID code 3ADK) and of the AK from Sulfolobus acidocaldarius, AKsa (PDB ID 1NKS-E).