Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine Rafael G. Silva a , Jose ´ H. Pereira b , Fernanda Canduri b , Walter F. de Azevedo Jr. b , Luiz A. Basso a, * , Dio ´genes S. Santos a, * a Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biome ´dicas, PUCRS, Porto Alegre, RS, Brazil b Departamento de Fı ´sica, UNESP, Sa ˜o Jose ´ do Rio Preto, SP, Brazil Received 2 May 2005, and in revised form 25 July 2005 Available online 18 August 2005 Abstract Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key posi- tions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Drug design; Purine nucleoside phosphorylase; MESG; Kinetic mechanism; Crystal structure Purine nucleoside phosphorylase (PNP) 1 (EC 2.4.2.1) is a key enzyme of the purine salvage pathway, respon- sible for the inter-conversion between (deoxy)nucleo- sides and bases, which in turn may be converted to uric acid for excretion or reused in nucleic acid biosyn- thesis [1]. This enzyme catalyzes the reversible cleavage, in the presence of inorganic phosphate (P i ), of N-ribos- idic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the corresponding purine base [2]. The reaction proceeds with inversion of configuration, from a-nucleosides to b- ribose 1-phosphate [3]. Human PNP (HsPNP) is classified, based on sub- strate specificity and structural characteristics, into the nucleoside phosphorylase-I family, which includes nucleoside phosphorylases with either trimeric or hexa- meric quaternary structure and a common single-do- main subunit, accepting both purine and pyrimidine nucleosides as substrates [4]. Interest in this enzyme has been greatly increased since the discovery that the congenital absence of its activity causes T-cell impair- ment in human beings, though keeping normal levels of B-cells [5,6]. Hence, HsPNP has being proposed as a target for drug design, aiming the enzyme inhibition 0003-9861/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.07.021 * Corresponding authors. Fax: +55 51 33203629. E-mail addresses: luiz.basso@pucrs.br (L.A. Basso), diogenes@ pucrs.br (D.S. Santos). 1 Abbreviations used: PNP, purine nucleoside phosphorylase; HsPNP, human purine nucleoside phosphorylase; P i , inorganic phos- phate; MESG, 7-methyl-6-thio-guanosine; bPNP, bovine purine nucleoside phosphorylase; V, maximal velocity; K m , Michaelis con- stant; k cat , catalytic constant; H-bond, hydrogen bond; K i , inhibition constant; Ino, inosine; ddI, 2 0 -3 0 -dideoxyinosine; ImmH, immucillin- H; ImmG, immucillin-G. Archives of Biochemistry and Biophysics 442 (2005) 49–58 www.elsevier.com/locate/yabbi ABB