Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine 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 substitution resulted in an 8-fold increase in Km for inosine from 58 to 484 μM and a 1000-fold decrease in kcat/Km. The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a Km of 45 μM and a kcat/Km 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in kcat/Km, 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in Km with inosine and 7-fold decrease in kcat/Km as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a Km of 42 μM and a kcat/Km twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 Å, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 Å for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation 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 the region of phosphate binding and the apparent S0.5 for phosphate with wild type and the Asn-243-Asp enzymes were 1.35±0.01 and 1.84±0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.
Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase: Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase
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Jason T. MAYNES, W.-S. YAM, Jack P. JENUTH, R. Gang YUAN, Steven A. LITSTER, Barry M. PHIPPS, Floyd F. SNYDER; Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase: Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase. Biochem J 1 December 1999; 344 (2): 585–592. doi: https://doi.org/10.1042/bj3440585
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