| Autor: |
Stoeckler JD; Section of Molecular & Biochemical Pharmacology, Division of Biology & Medicine, Brown University, Providence, Rhode Island 02912, USA., Poirot AF, Smith RM, Parks RE Jr, Ealick SE, Takabayashi K, Erion MD |
| Abstrakt: |
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. |
