Design of an adenosine phosphorylase by active-site modification of murinepurine nucleoside phosphorylase - Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase

Our objective was to alter the substrate specificity of purine nucleoside p hosphorylase such that it would catalyse the phosphorolysis of 6-aminopurin e nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine, The Asn-243-Asp substitutio n resulted in an 8-fold increase in K-m for inosine from 58 to 484 mu M and a 1000-fold decrease in k(cat)/K-m. The Asn-243-Asp construct catalysed th e phosphorolysis of adenosine with a K-m of 45 mu M and a k(cat)/K-m 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reductio n in k(cat)/K-m, 83% of wild type, but had no activity with adenosine, The Asn-243-Asp; Lys-244-Gln construct had a 14-fold increase in K-m with inosi ne and 7-fold decrease in k(cat)/K-m as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K-m of 42 mu M and a k(cat)/K-m twice that of the single Asn-243-Asp substitution. Molec ular dynamics simulation of the engineered proteins with adenine as substra te revealed favourable hydrogen bond distances between N7 of the purine rin g and the Asp-243 carboxylate at 2.93 and 2.88 Angstrom, for Asn-243-Asp an d the Asn-243-Asp; Lys-244-Gln constructs respectively. Simulation also sup ported a favourable hydrogen bond distance between the purine C6-amino grou p and Asp-243 at 2.83 and 2.88 Angstrom for each construct respectively, Th e Asn-243-Thr substitution did not yield activity with adenosine and simula tion gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in th e region of phosphate binding and the apparent S-0.5 for phosphate with wil d type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.66 mM, res pectively. Both proteins exhibited positive co-operativity with phosphate g iving Hill coefficients of 7.9 and 3.8 respectively.