Insight into catalytically relevant correlated motions in human purine nucleoside phosphorylase.
The catalytic site of the homotrimeric enzyme human purine nucleoside phosphorylase enzyme (hPNP) features residue F200 and the 241-265 loop directly skirting the purine base and a residue belonging to the adjacent monomer, F159, immediately conterminous to the ribosyl moiety. Crystallographic B-factors of apo human purine nucleoside phosphorylase, and hPNP complexed with substrate/transition state (TS) analogues, show that residue E250 is the centroid of a highly mobile loop region. Furthermore, superimposition of apo hPNP and hPNP complexed with TS analogue Immucillin-H shows a tightening of the active site, caused by the ligand-dependent 241-265 loop rearrangement taking place upon substrate/inhibitor binding, suggesting a putative dynamic role of the loop in binding/catalysis. However, crystallographic structures reveal only average atomic positions, and more detailed information is needed to discern the dynamic behavior of hPNP. The Essential Dynamics (ED) method is used here to investigate the existence of correlated motions in hPNP and consequently proposes mutagenesis assays to estimate the relative importance of these motions in the phosphorolytic efficiency of the reaction catalyzed by hPNP. We compare the concerted motions obtained from multiple molecular dynamics simulations of apo and Michaelis complex of hPNP both in vacuo and in solution. The results of the principal component analysis for the apo hPNP indicate the existence of strong correlations predominantly in the vicinity of residue F159. However, for the Michaelis complex, concerted motions are seen mostly around both active site residue F200 and loop residue E250. Additionally, for a simulation depicting the relaxation of tight complexed hPNP with a TS analogue, toward its relaxed apo form (after removal of the TS analog), a combination of the apo hPNP and Michaelis complex motions is found, with prominent concerted modes centered around neighboring residues F159, F200, and E250. Finally, we probed the extent to which these concerted motions bear an intrinsic catalytic role by performing experimental site-directed mutagenesis on some residues, followed by kinetic analysis. The F159G and F200G mutants displayed a strong increase in K(M) and modest decrease in k(cat), suggesting that these concerted motions may provide dynamical roles in substrate binding and/or catalysis. However, further structural data for the hPNP mutants are needed to confirm our hypothesis.
Núñez
Sara
S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wing
Corin
C
Antoniou
Dimitri
D
Schramm
Vern L
VL
Schwartz
Steven D
SD
D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D009038:Motion; D016297:Mutagenesis, Site-Directed; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics
D011683:Purine-Nucleoside Phosphorylase