Purine Nucleoside Phosphorylase. Transition State Structure, Transition State Inhibitors and One-Third-The-Sites Reactivity

摘要

The genetic deficiency of purine nucleoside phosphorylase (PNP) in humans causes T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. The transition state structure for PNP has been solved by kinetic isotope effect methods for both the arsenolysis reaction and a PNP hydrolytic reaction and is characterized by 1) an elevated pK_a at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and 2) oxocarbenium ion formation in the ribosyl ring and 3) minimal participation of the nucleophile at the transition state (Kline, P. C, & Schramm, V. L. (1995) Biochemistry 34, 1153-1162). These features were considered in the design of stable transition state analogues; l,4-dideoxy-4-imino-l-(9-deazahypoxanthine)-D-ribitol (immucillin-H) and l,4-dideoxy-4-imino-l-(9-deazaguanine)-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylases. The equilibrium dissociation constants (Ki) are 23 to 72 pM, making these the most powerful inhibitors reported for the enzyme. The similarity of the molecular electrostatic potential surfaces of the transition state and the transition state inhibitors account for the transition state inhibitor affinity. Complete inhibition of the homotrimeric enzymes occurs with one mole of inhibitor per mole of enzymic homotrimer. An inhibitor molecule bound tightly at one site prevents binding at the two remaining homotrimer sites. In the absence of phosphate, the enzyme catalyzes a one-third-the-sites hydrolysis of inosine and forms a tightly bound PNPhypoxanthine complex at one of the three catalytic sites (Kd = 1.3 pM). The remaining sites are inactive until the hypoxantnine is released. Ground-state inhibitors (substrate and product analogues) bind equally to all three homotrimer subunits. A mechanism of sequential subunit catalysis, with tightly bound product, reminiscent of the mechanism of F_1 ATPase, is indicated by these results. The transition state inhibitors and inosine hydrolysis capture part of the transition state energy which is proposed to occur sequentially, one subunit at a time, in the normal catalytic cycle. Large global conformational changes occur when transition state inhibitors are bound at one site of the homotrimeric PNP. The inhibitors have potential as biologically active agents.