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.
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 K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction 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 inosine 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 microM and a k(cat)/K(m) 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 A, 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 A 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 S(0.5) 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.
Maynes
J T
JT
Departments of Medical Genetics Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
Yam
W
W
Jenuth
J P
JP
Gang Yuan
R
R
Litster
S A
SA
Phipps
B M
BM
Snyder
F F
FF
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D003198:Computer Simulation; D005573:Formycins / Q000494:pharmacology; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D005573:Formycins; D005973:Glutamine; C034561:formycin B; D001224:Aspartic Acid; C034550:formycin; D001216:Asparagine; C019603:adenosine phosphorylase; D011683:Purine-Nucleoside Phosphorylase; D008239:Lysine