Reversal of ADP-Mediated Aggregation of Adenosine Kinase by Cyclophilin Leads to Its Reactivation ² Banibrata Sen, ‡ Anutosh Chakraborty, ‡,§ Rupak Datta, ‡ Debasish Bhattacharyya, | and Alok K. Datta* ,‡ Leishmania Group, DiVision of Infectious Diseases, and Department of Drug Design, DeVelopment and Molecular Modeling, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India ReceiVed September 12, 2005; ReVised Manuscript ReceiVed October 27, 2005 ABSTRACT: Cyclophilins have been implicated in several important cellular functions. Our earlier results showed that reactivation of adenosine kinase (AdK) by CyP (LdCyP) from the parasitic protozoa Leishmania donoVani is accompanied with disaggregation of the enzyme [Chakraborty, A., et al. (2002) J. Biol. Chem. 277, 47451-47460; Chakraborty, A., et al. (2004) Biochemistry 43, 11862-11872]. However, it remained to be known why the enzyme displayed progressive inhibition during the time-dependent reaction and what LdCyP does to prevent and/or reverse the inhibition. Herein, we demonstrate that one of its reaction products, ADP but not AMP, facilitates the formation of AdK aggregates, leading to its inactivation. Further studies revealed that LdCyP reactivates the enzyme by withdrawing the ADP inhibition. To investigate the molecular mechanism, the intrinsic tryptophan fluorescence and polarization of AdK were monitored in the presence of either LdCyP or ADP and in combination thereof. Whereas in the presence of LdCyP the tryptophan fluorescence emission maxima of AdK exhibited a red shift, ADP had a quenching effect. However, both the red shift and quenching became less noticeable when one (W234) of the two tryptophan residues of AdK was altered, indicating W234 fluorescence is relatively more sensitive to both LdCyP and ADP binding. Kinetic measurements indicated that LdCyP-facilitated reactivation of AdK is accompanied with a concomitant increase in the K D of ADP but not of AMP. Interestingly, addition of myokinase (MK) and pyruvate kinase (PK) along with phosphoenolpyruvate, either singly or in conjunction, to the AdK reaction mixture led to its reactivation. The effect of PK but not of MK could be substituted by CyP and vice versa. Taken together, the results suggest that LdCyP-induced reactivation occurs due to conformational reorientation of AdK in a manner that decreases the affinity of the enzyme for ADP with consequent relief from the ADP-mediated aggregation. Cyclophilins (CyPs), 1 a multigenic family of ubiquitous proteins carrying peptidyl-prolyl cis-trans isomerase (PPI- ase) activity, are the specific receptors of the immunosup- pressive drug cyclosporin A (CsA) (1, 2). Besides mediating the CsA-induced immunosuppression, CyPs are involved in various cellular processes that span from cell division, receptor maturation, acceleration of protein folding, and protection against human immunodeficiency virus (HIV) infection to several other processes (3-13). Alterations of activities of various enzymes and transcription factors, following interaction with CyP, have also been reported ( 14- 16). In another report, it has been shown that CyP keeps peroxiredoxin proteins activated by keeping the enzyme in the reduced state (17). Leishmania donoVani, a dimorphic purine auxotrophic parasitic protozoon, is the causative agent for human kala- azar. This parasite exists as a flagellated promastigote (extracellular form) in the sand fly vector and is transformed into amastigote (intracellular form) in the mammalian macrophages. During the process of transformation, the activity of a large number of proteins has been reported to be stage-specifically altered (18, 19). Adenosine kinase (AdK), one of the purine salvage pathway enzymes, which by following a sequential bi-bi orderly mechanism carries out phosphorylation of Ado in the presence ATP to form AMP and ADP, plays a pivotal role in the metabolism and uptake of Ado, a key modulator of various cellular functions (20, 21). Moreover, the fact that the activity of this enzyme from all known sources is regulated by its substrates and reaction products has been known for a long time (22-25). In L. donoVani, the enzyme has been shown to display several-fold higher activity upon transformation into amastigotes (26). However, to date, no evidence exists to indicate that the higher activity observed was due to an acceleration of protein synthesis. ² This work was supported by grants from the Department of Science & Technology (SP/SO/D-38/2000) and CSIR Network Project (SMM003), Government of India. B.S. and R.D. were each supported by individual fellowships from the Council of Scientific & Industrial Research, Government of India. * To whom correspondence should be addressed. Telephone: +91 33 2473-6793. Fax: +91 33 2473-5197. E-mail: alokdatta@iicb.res.in. ‡ Division of Infectious Diseases. § Present address: Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. | Department of Drug Design, Development and Molecular Modeling. 1 Abbreviations: DTT, dithiothreitol; PK, pyruvate kinase; MK, myokinase; AdK, adenosine kinase; CsA, cyclosporin A; LdCyP, Leishmania donoVani cyclophilin; CyP, cyclophilin; Ado, adenosine; PEP, phosphoenolpyruvate; FKBP, FK506 binding protein; Ni-NTA, nickel-nitrilotriacetic acid. 263 Biochemistry 2006, 45, 263-271 10.1021/bi0518489 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/06/2005