Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitorHPP reveals the involvement of the flexible loop in substrate binding

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthi ne phosphoribosyltransferase (HGPRTase) with alanine to exploit this less r eactive form of the enzyme to gain additional insights into the structure a ctivity relationship of HGPRTase. Although this substitution resulted in on ly a minimal (one- to threefold) increase in the K-m values for binding pyr ophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(ca t)/K-m) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in bindin g of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K6 8A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyr azolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(F-0 - F-c)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyropho sphate binding domain as well as in a long flexible loop (residues Leu101 t o Gly111) that closes over the active site. Loop closure reveals the functi onal roles for the conserved SY dipeptide of the loop as well as the molecu lar basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.