1.45 A resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Journal Article · Fri Jan 01 04:00:00 EST 2010 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2009.11.124· OSTI ID:22199983

Chojnowski, Grzegorz ^[1]; Breer, Katarzyna; Narczyk, Marta; Wielgus-Kutrowska, Beata ^[1]; Czapinska, Honorata ^[2]; Hashimoto, Mariko; Hikishima, Sadao; Yokomatsu, Tsutomu ^[3]; Bochtler, Matthias ^[2]; Girstun, Agnieszka; Staron, Krzysztof ^[4]; Bzowska, Agnieszka ^[1]

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1. Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw (Poland)
2. International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland)
3. School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)
4. Department of Molecular Biology, Institute of Biochemistry, University of Warsaw, Miecznikowa 1, 02-096 Warsaw (Poland)

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K{sub d} ({approx}190 pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45 A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

• OSTI ID: 22199983
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 391; ISSN BBRCA9; ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
CALVES
CRYSTAL STRUCTURE
CRYSTALLOGRAPHY
DISSOCIATION
ENZYME ACTIVITY
GUANINE
HYPOXANTHINE
INHIBITION
INOSINE
LIGANDS
PHOSPHOTRANSFERASES
PIPERAZINES
RIBOSE
TITRATION