Limits...
Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Wildberger P, Aish GA, Jakeman DL, Brecker L, Nidetzky B - Biochem Biophys Rep (2015)

Bottom Line: Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase.Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested.High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed (4) C 1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp(196), Glu(237) and Asp(295)) and from Arg(137). Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe(52) by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

No MeSH data available.


Free-energy profile comparison for the catalytic reaction of F52A_D295A mutant (gray) with catalytic reactions of wild-type LmSPase (black dashed), F52A mutant (blue) and D295N (green) mutant. A standard state of 1 M was assumed. Kinetic parameters are from Table S2 in the Supporting Information. An equilibrium constant Keq of 9 was used (pH 7.0, 30 °C). The free-energy profiles were constructed as described elsewhere [11]. Underlying equations are furthermore provided in Table S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4554294&req=5

f0015: Free-energy profile comparison for the catalytic reaction of F52A_D295A mutant (gray) with catalytic reactions of wild-type LmSPase (black dashed), F52A mutant (blue) and D295N (green) mutant. A standard state of 1 M was assumed. Kinetic parameters are from Table S2 in the Supporting Information. An equilibrium constant Keq of 9 was used (pH 7.0, 30 °C). The free-energy profiles were constructed as described elsewhere [11]. Underlying equations are furthermore provided in Table S2.

Mentions: Detailed steady-state kinetic characterization of F52A_D295A double mutant was performed. The resulting kinetic parameters are summarized in Table S2 along with kinetic parameters of the wild-type enzyme, F52A [11] and D295N mutant [9] that were determined in earlier studies. The two-step reaction mechanism of LmSPase (Scheme 1) implies that the kcat/Km for glucosyl donor (sucrose, αGlc 1-P) and acceptor (phosphate, d-fructose) is the relevant kinetic expression for glucosylation and deglucosylation of Asp196 in the direction of phosphorolysis and synthesis, respectively. Fig. 3 compares the free-energy profiles constructed from the kcat/Km values assuming a 1 M standard state. Kinetic consequences in F52A_D295A mutant are discussed based on differences in free energy compared to the other phosphorylases.


Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Wildberger P, Aish GA, Jakeman DL, Brecker L, Nidetzky B - Biochem Biophys Rep (2015)

Free-energy profile comparison for the catalytic reaction of F52A_D295A mutant (gray) with catalytic reactions of wild-type LmSPase (black dashed), F52A mutant (blue) and D295N (green) mutant. A standard state of 1 M was assumed. Kinetic parameters are from Table S2 in the Supporting Information. An equilibrium constant Keq of 9 was used (pH 7.0, 30 °C). The free-energy profiles were constructed as described elsewhere [11]. Underlying equations are furthermore provided in Table S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554294&req=5

f0015: Free-energy profile comparison for the catalytic reaction of F52A_D295A mutant (gray) with catalytic reactions of wild-type LmSPase (black dashed), F52A mutant (blue) and D295N (green) mutant. A standard state of 1 M was assumed. Kinetic parameters are from Table S2 in the Supporting Information. An equilibrium constant Keq of 9 was used (pH 7.0, 30 °C). The free-energy profiles were constructed as described elsewhere [11]. Underlying equations are furthermore provided in Table S2.
Mentions: Detailed steady-state kinetic characterization of F52A_D295A double mutant was performed. The resulting kinetic parameters are summarized in Table S2 along with kinetic parameters of the wild-type enzyme, F52A [11] and D295N mutant [9] that were determined in earlier studies. The two-step reaction mechanism of LmSPase (Scheme 1) implies that the kcat/Km for glucosyl donor (sucrose, αGlc 1-P) and acceptor (phosphate, d-fructose) is the relevant kinetic expression for glucosylation and deglucosylation of Asp196 in the direction of phosphorolysis and synthesis, respectively. Fig. 3 compares the free-energy profiles constructed from the kcat/Km values assuming a 1 M standard state. Kinetic consequences in F52A_D295A mutant are discussed based on differences in free energy compared to the other phosphorylases.

Bottom Line: Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase.Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested.High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed (4) C 1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp(196), Glu(237) and Asp(295)) and from Arg(137). Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe(52) by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

No MeSH data available.