Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues.
Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.
Rinaldo-Matthis
Agnes
A
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wing
Corin
C
Ghanem
Mahmoud
M
Deng
Hua
H
Wu
Peng
P
Gupta
Arti
A
Tyler
Peter C
PC
Evans
Gary B
GB
Furneaux
Richard H
RH
Almo
Steven C
SC
Wang
Ching C
CC
Schramm
Vern L
VL
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives; D000818:Animals; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011759:Pyrrolidines / Q000737:chemistry* / Q000494:pharmacology*; D013379:Substrate Specificity; D014246:Trichomonas vaginalis / Q000201:enzymology
C517547:4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillin A; D011759:Pyrrolidines; D011683:Purine-Nucleoside Phosphorylase; D000225:Adenine; D000241:Adenosine; C517546:galidesivir