Journal of Biomolecular NMR, 13: 195–196, 1999. KLUWER/ESCOM © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 195 Letter to the Editor: Sequence-specific 1 H, 15 N and 13 C assignment of adenylate kinase from Escherichia coli in complex with the inhibitor AP 5 A Eva Meirovitch ∗ , Michael A. Sinev & Elena V. Sineva Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Received 20 August 1998; Accepted 7 October 1998 Key words: adenylate kinase, dynamic properties and enzyme function, NMR assignment Biological context Adenylate kinase (AK) is a small multi-domain monomeric enzyme catalyzing the transfer of a phos- phoryl group from ATP to AMP. Crystallographic studies on AKs suggest closure of enzyme domains upon formation of the enzyme–substrate ternary com- plex (Vonrhein et al., 1995). Time-resolved fluorescence energy-transfer stud- ies of E. coli AK (AKeco) revealed that, unlike the pseudo-ternary complex AKeco ∗ AP 5 A [P 1 ,P 5 - di(adenosine-5 ′ )pentaphosphate], the ligand-free en- zyme features substantial inter-domain flexibility (Sinev et al., 1996). The latter data address the role of dynamic phenomena in AK catalysis. The nearly com- plete sequence-specific assignment of AKeco ∗ AP 5 A provides the basis for elucidating dynamic and struc- tural properties that control kinase catalysis. It should be noted that 1 H, 15 N and 13 C assign- ments and the secondary structure of an AK variant from chicken muscle in complex with AP 5 A have been published by Byeon et al. (1993). A set of (mainly backbone) 1 H assignments of AKeco were determined recently by Burlacu-Miron et al. (1998). Methods and results Recombinant plasmid pEAK91, containing the intact gene coding for E. coli AK (Reinstein et al., 1988), was a gift of Prof. Wittinghofer (Max-Planck Insti- tute for Molecular Physiology, Dortmund, Germany). For the preparation of 15 N, 13 C-labeled AKeco, E. coli HB101 cells, transformed with the pEAK91 plasmid, were grown at 37 ◦ C in Celtone-CN medium (Martek ∗ To whom correspondence should be addressed. Biosciences Corp.). The protein was purified as de- scribed previously (Sinev et al., 1996). The yield of purified enzyme was about 110 mg per liter of cell culture. AKeco stock solutions were prepared in 40 mM sodium-phosphate buffer (pH 6.8). To prepare the 100% D 2 O sample, 290 µl of the AKeco ∗ AP 5 A so- lution in sodium phosphate buffer was freeze-dried, dissolved in 400 µl of D 2 O, incubated for 24 h at room temperature, freeze-dried again, and dissolved in D 2 O to a final volume of 290 µl. AKeco/AP 5 A concen- trations were 2.7 mM/5.7 mM (2.1 mM/5.6 mM) in the 95% H 2 O/5% D 2 O (100% D 2 O) sample. Sample volumes of 250 µl were used in Shigemi cells. 15 N-HSQC, 13 C-HSQC, 3D CBCA(CO)NH, 3D HNCACB, 3D HNCO, 3D HBHA(CBCACO)NH, 3D C(CO)NH, 3D H(CCO)NH, 3D HCCH-TOCSY, 3D HCCH-COSY, 3D 15 N-edited NOESY, and 3D 13 C- edited NOESY experiments (Bax and Grzesiek, 1993) were carried out at 303 K on a Bruker DMX 600 MHz spectrometer, in the phase-sensitive mode. Pulse se- quences developed by Bax and co-workers, with typ- ical acquisition parameters as outlined in the original papers, were used. The spectra were processed using NMRDraw/NMRPipe (Delaglio et al., 1995). Forward (mirror-image) linear prediction was used for semi- constant (constant) indirect evolution time periods. Apodization with a cosine-bell function in the acqui- sition dimension, and a cosine function in the indirect dimensions, was applied. The time-domain data were zero-filled to the next power of two. The 15 N-HSQC plane contained 202 out of the 203 expected δ( 1 H N i ), δ( 15 N i ) correlations. Establishing sequential connectivity among these coordinates, and determining the respective amino acid type, was car- ried out simultaneously using 3D HNCACB and 3D