PURINE NUCLEOSIDE PHOSPHORYLASE .3. REVERSAL OF PURINE BASE SPECIFICITY BY SITE-DIRECTED MUTAGENESIS

Human purine nucleoside phosphorylase (PNP) is highly specific for 6-o xopurine nucleosides with a catalytic efficiency (k(cat)/K-M) for inos ine 350000-fold greater than for adenosine. Crystallographic studies i dentified Asn243 and Glu201 as the residues largely responsible for th e substrate specificity. Results from mutagenesis studies demonstrated that the side chains for both residues were also essential for effici ent catalysis [Erion, M. D., et al. (1997a) Biochemistry, 36, 11725-11 734], Additional mechanistic studies predicted that Asn243 participate d in catalysis by stabilizing the transition state structure through h ydrogen bond donation to N7 of the purine base [Erion, M. D., et al. ( 1997b) Biochemistry 36, 11735-11748]. In an effort to alter the substr ate specificity of human PNP, mutants of Asn243 and Glu201 were design ed to reverse hydrogen bond donor and acceptor interactions with the p urine base. Replacement of Asn243 with Asp, but not with other amino a cids, led to a 5000-fold increase in k(cat) for adenosine and a 4300-f old increase in overall catalytic efficiency, Furthermore, the Asn243A sp mutant showed a 2.4-fold preference for adenosine relative to inosi ne and a 800000-fold change in substrate specificity (k(cat)/K-M) rela tive to wild-type PNP. The double mutant, Asn243Asp::Glu201Gln, exhibi ted a 190-fold increase in catalytic efficiency with adenosine relativ e to wild-type PNP, a 480-fold preference for adenosine relative to in osine, and a 1.7 x 10(8)-fold change in preference for adenosine over inosine relative to wildtype 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 m utants represent examples in which protein engineering significantly a ltered substrate specificity while maintaining high catalytic efficien cy.