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. 2008 May 15;94(10):4078-88.
doi: 10.1529/biophysj.107.121913. Epub 2008 Jan 30.

Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase

Suwipa Saen-Oon  1 , Mahmoud Ghanem, Vern L Schramm, Steven D Schwartz
Affiliations

Remote mutations and active site dynamics correlate with catalytic properties of purine nucleoside phosphorylase

Suwipa Saen-Oon et al. Biophys J. .

Abstract

It has been found that with mutation of two surface residues (Lys(22) --> Glu and His(104) --> Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5'...ImmG:N4' and H257:Ndelta...ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'...ImmG:O5'...H257:Ndelta became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

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Figures

FIGURE 1
FIGURE 1
PNP-catalyzed phosphorolysis of guanosine yielding guanine and α-d-ribose 1-phosphate. ImmG is a TS analog mimic of the TS oxacarbenium ion character and is a picomolar inhibitor of human PNP.
FIGURE 2
FIGURE 2
(a) Asymmetric unit of trimer hPNP with the catalytic site of subunit-A located near the trimer interface and covered with the loop including Phe-159C of subunit C. Mutated residues His104Arg and Lys22Glu are located 25 Å and 45 Å away from the catalytic site of the adjacent subunit and 26 Å and 32 Å from the catalytic site of their own subunit. (b) The active site residues near ImmG and the phosphate nucleophile. (c) Structure of ImmG at the catalytic site of hPNP. Active site residues in contact with ImmG and the nucleophile phosphate are shown. The ImmG:O5′, ImmG:N4′, H257:Nδ, and formula image:Op atoms are highlighted in red.
FIGURE 3
FIGURE 3
(a) Superposition of trimeric bvPNP complexed with acyclic nucleoside phosphonate inhibitor (2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine) (pdb code 1LVU, blue) and the modeled trimer of hPNP complexed with ImmH·formula image (pdb code 1RR6, orange). (b) Superposition of the crystal structure of monomeric bvPNP (ribbon enzyme blue; stick ImmH·formula image cyan) with hPNP (ribbon enzyme orange; stick ImmH·formula image red).
FIGURE 4
FIGURE 4
Reaction scheme of PNP (E) for conversion of guanosine to guanine and ribose 1-phosphate (R1-P). Only the enzyme-bound guanine is fluorescent under these conditions. Product release (k7) is rate limiting.
FIGURE 5
FIGURE 5
RMSD of the backbone atoms (a) and radius of gyration (b) of the trimeric complexes of hPNP·ImmG·formula image and E:R hPNP·ImmG·formula image in the last 100 ps of 260 ps molecular dynamic simulations.
FIGURE 6
FIGURE 6
Power spectra of the Op⋯N4′ distance autocorrelation function and the Op⋯N4′ and O5′⋯N4′ distance-distance cross-correlation functions of hPNP·ImmG·formula image (a) and E:R hPNP·ImmG·formula image (b).
FIGURE 7
FIGURE 7
Power spectra of the O5′⋯N4′ distance autocorrelation function and the O5′⋯N4′ and H257:Nδ⋯O5′ distance-distance cross-correlation functions of hPNP·ImmG·formula image (a) and E:R hPNP·ImmG·formula image (b).
FIGURE 8
FIGURE 8
Power spectra of the O5′⋯O4′ distance autocorrelation function and the O5′⋯O4′ and H257:Nδ⋯O5′ distance-distance cross-correlation functions of guanosine and formula image complexed with hPNP and E:R hPNP (a). The plots show the distance O5′⋯O4′, H257:Nδ⋯O5′, and the summation of two distances throughout the last 100 ps of a 260 ps simulation (b).
FIGURE 9
FIGURE 9
Residue flexibility for backbone atoms of hPNP (a) and E:R hPNP (b). Ribbon diagrams (c and d) are colored on the basis of the relative amplitudes of fluctuations of individual residue for hPNP and E:R hPNP, respectively. A blue-to-red color spectrum is used to represent different levels of flexibilities, where the smallest motions are in blue and the largest are in red. The phosphate-loop and Phe-159C-loop regions are indicated. Structure of ImmG, His-257A, Phe-200A, Tyr-88A, and Phe-159C are shown.
FIGURE 10
FIGURE 10
Eigenvectors 1–3 (a–c) of hPNP and E:R hPNP in complex with ImmG and phosphate. The collective motions of subunit A (black), subunit B (red), and subunit C (green) are indicated.
FIGURE 11
FIGURE 11
Details of the ImmG (green) binding site in subunit A of hPNP (a) and E:R hPNP (b). The top panel shows the side chain of Arg-104C (magenta) perturbing the flexibility (dynamics) of the Phe-159C loop (red) to increase its mobility, represented by the color spectrum of backbone fluctuation. The Phe-159C (red) contacting the catalytic site alters the dynamics of the residues His-257A, Phe-200A, and Tyr-88A (orange), which form a hydrophobic cluster. Those hydrophobic residues pack in a more favorable conformation in E:R hPNP. The yellow arrows (b) indicate the vector of conformational perturbation. The average structure of the last 100 ps of a total 260 ps dynamic simulation is shown.
FIGURE 12
FIGURE 12
Average distances between residues (a) and pairwise nonbonded interaction energies (b) averaged over 1000 structures during the last 100 ps of a total 260 ps dynamic simulation (0.1 ps configurations sampling). The error bars show the standard deviations.
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References

    1. Garcia-Viloca, M., J. Gao, M. Karplus, and D. G. Truhlar. 2004. How enzymes work: analysis by modern rate theory and computer simulations. Science. 303:186–195. - PubMed
    1. Thorpe, I. F., and C. L. Brooks. 2005. Conformational substates modulate hydride transfer in dihydrofolate reductase. J. Am. Chem. Soc. 127:12997–13006. - PubMed
    1. Bruice, T. C. 2006. Computational approaches: reaction trajectories, structures, and atomic motions. Enzyme reactions and proficiency. Chem. Rev. 106:3119–3139. - PubMed
    1. Pu, J. Z., J. Gao, and D. G. Truhlar. 2006. Multidimensional tunneling, recrossing, and the transmission coefficient for enzymatic reactions. Chem. Rev. 106:3140–3169. - PMC - PubMed
    1. Antikainen, N. M., R. D. Smiley, S. J. Benkovic, and G. G. Hammes. 2005. Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase. Biochemistry. 44:16835–16843. - PubMed

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