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.
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December 1999
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Research Article|
November 24 1999
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
Jason T. MAYNES;
Jason T. MAYNES
*Departments of Medical Genetics and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
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W.-S. YAM;
W.-S. YAM
*Departments of Medical Genetics and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
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Jack P. JENUTH;
Jack P. JENUTH
1
*Departments of Medical Genetics and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
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R. Gang YUAN;
R. Gang YUAN
*Departments of Medical Genetics and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
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Steven A. LITSTER;
Steven A. LITSTER
2
†Department of Biological Science, Faculty of Science, University of Calgary, 2500 University Drive N.W., Calgary, Alberta T2N 1N4, Canada
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Barry M. PHIPPS;
Barry M. PHIPPS
†Department of Biological Science, Faculty of Science, University of Calgary, 2500 University Drive N.W., Calgary, Alberta T2N 1N4, Canada
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Floyd F. SNYDER
Floyd F. SNYDER
3
*Departments of Medical Genetics and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada
1To whom correspondence should be addressed (e-mail snyder@ucalgary.ca).
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Publisher: Portland Press Ltd
Received:
April 22 1999
Revision Received:
August 16 1999
Accepted:
September 10 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 344 (2): 585–592.
Article history
Received:
April 22 1999
Revision Received:
August 16 1999
Accepted:
September 10 1999
Citation
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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