mechanism: 1. random binding of substrates, 2. potent binding and slow release of some reaction products leading to the circumstances that the chamical step is not the slowest one and that rapid-equilibrium assumptions do not hold, 3. dual role of phosphate, as substrate and as reaction modifier
steady-state ordered bi bi kinetic mechanism, in which phosphate binds first followed by 2'-deoxyguanosine, and ribose 1-phosphate dissociates first followed by guanine. A general acid is essential for both catalysis and 2'-deoxyguanosine binding, and deprotonation of a group abolishes phosphate binding. Product release contributes to the rate-limiting step
the structure of hPNP is a homotrimer with the catalytic sites near the subunit interfaces. The ternary complex of hPNP includes the binding of inosine and hydrogen phosphate to the active site. The reaction consists of a phosphorolysis at C1' of inosine's ribose with inversion of stereochemistry at this position. Reaction occurs via an SN1-like mechanism where hydrogen phosphate nucleophile and the purine base leaving group are separated from the oxocarbenium ion, defining a dissociative transition state. It has to be pointed out that the reaction involves annihilation of charges in going from the reactant state to the product state. Reaction mechanism analysis, quantum mechanical and molecular mechanical (QM/MM) molecular dynamics (MD) simulations, detailed overview
the trimeric PNPs show that there is no acidic residue in the vicinity of the purine ring N7, only the side-chain of Asn243 (Asn246 in Cellulomonas PNP) is found there. Moreover, in the latter structure, Asn246 interacts with purine through a water molecule, questioning the protonation mechanism in the catalysis. The molecular mechanism of catalysis of trimeric PNPs involves either protonation of the purine ring N7, or a negatively charged purine intermediate stabilized by hydrogen bonds of purine N(7) with Asn243, or of the purine N1H with Glu201 (Glu204 in Cellulomonas PNP). Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. The alternative mechanism assumes a negatively charged purine ring in the transition state stabilized by a hydrogen bond from Asn243 to purine ring N7. Key catalytic role of Glu204
the trimeric PNPs show that there is no acidic residue in the vicinity of the purine ring N7, only the side-chain of Asn243 is found there. The molecular mechanism of catalysis of trimeric PNPs involves either protonation of the purine ring N7, or a negatively charged purine intermediate stabilized by hydrogen bonds of purine N(7) with Asn243, or of the purine N1H with Glu201. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. The alternative mechanism assumes a negatively charged purine ring in the transition state stabilized by a hydrogen bond from Asn243 to purine ring N7. Key catalytic role of Glu201
the trimeric PNPs show that there is no acidic residue in the vicinity of the purine ring N7, only the side-chain of Asn243 is found there. The molecular mechanism of catalysis of trimeric PNPs involves either protonation of the purine ring N7, or a negatively charged purine intermediate stabilized by hydrogen bonds of purine N(7) with Asn243, or of the purine N1H with Glu201. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. The alternative mechanism assumes a negatively charged purine ring in the transition state stabilized by a hydrogen bond from Asn243 to purine ring N7. Key catalytic role of Glu201