Purine Nucleoside Phosphorylase. 3. Reversal of Purine Base Specificity by Site-Directed Mutagenesis

Abstract

Human purine nucleoside phosphorylase (PNP) is highly specific for 6-oxopurine nucleosides with a catalytic efficiency (kcat/KM) for inosine 350000-fold greater than for adenosine. Crystallographic studies identified Asn243 and Glu201 as the residues largely responsible for the substrate specificity. Results from mutagenesis studies demonstrated that the side chains for both residues were also essential for efficient catalysis [Erion, M. D., et al. (1997a) Biochemistry 36, 11725-11734]. Additional mechanistic studies predicted that Asn243 participated in catalysis by stabilizing the transition state structure through hydrogen bond donation to N7 of the purine base [Erion, M. D., et al. (1997b) Biochemistry 36, 11735-11748]. In an effort to alter the substrate specificity of human PNP, mutants of Asn243 and Glu201 were designed to reverse hydrogen bond donor and acceptor interactions with the purine base. Replacement of Asn243 with Asp, but not with other amino acids, led to a 5000-fold increase in kcat for adenosine and a 4300-fold increase in overall catalytic efficiency. Furthermore, the Asn243Asp mutant showed a 2.4-fold preference for adenosine relative to inosine and a 800000-fold change in substrate specificity (kcat/KM) relative to wild-type PNP. The double mutant, Asn243Asp::Glu201Gln, exhibited a 190-fold increase in catalytic efficiency with adenosine relative to wild-type PNP, a 480-fold preference for adenosine relative to inosine, and a 1.7 x 10(8)-fold change in preference for adenosine over inosine relative to wild-type PNP. The Asn243Asp mutant was also shown to synthesize 2,6-diaminopurine riboside with a catalytic efficiency (1.4 x 10(6) M-1 s-1) on the same order of magnitude as wild-type PNP with its natural substrates hypoxanthine and guanine. The Asn243Asp mutants represent examples in which protein engineering significantly altered substrate specificity while maintaining high catalytic efficiency.

YLR209c Encodes Saccharomyces cerevisiae Purine Nucleoside Phosphorylase

“…3B). Guanosine can therefore be bound more efficiently than inosine by yeast PNP, a situation similar to that reported for human PNP for which the K m s for guanosine and inosine are 12 and 45 M, respectively (17). Finally, the V max and k cat of PNP, with inosine as substrate, were 2.6 M min Ϫ1 and 38 s Ϫ1 , respectively.…”

“…Finally, the V max and k cat of PNP, with inosine as substrate, were 2.6 M min Ϫ1 and 38 s Ϫ1 , respectively. This latter parameter of the yeast enzyme is therefore similar to the one reported for human PNP and inosine: 57 s Ϫ1 for k cat (17). A pnp1 null mutant (purchased from Euroscarf) did not show any obvious growth defect.…”

Three-dimensional Structure of a Hyperthermophilic 5′-Deoxy-5′-methylthioadenosine Phosphorylase from Sulfolobus solfataricus

“…In addition to differences in quaternary structure, it has been shown that in general, the mammalian trimeric PNPs have a higher substrate specificity and efficiency than the hexameric enzymes (6,30,31). The crystal structures of these enzymes together with biochemical and mutational studies (29,32,33) have allowed detailed mapping of the active sites for several enzymes in the PNP family and specific roles in substrate binding and catalysis have been assigned. While all of these enzymes utilize inorganic phosphate to cleave the glycosidic bond of nucleosides, key structural differences exist which account for the variation in substrate specificities.…”

Purine nucleoside phosphorylase

“…Скорость фосфоролиза и/или синтеза аденозина на 1% меньше скорости таковой для инозина [70]. Однако, некоторые ПНФ конкурентно ингибируются аденином и/или аденозином, а, следовательно, связывают их, как например, ПНФ из клеток саркомы 180, Cellulomonas, Proteus vulgaris [67].…”