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Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.

Pearson D, Carell T - Nucleic Acids Res. (2011)

Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations.This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

ABSTRACT
Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.

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HPLCs and Michaelis–Menten plots for each MnmC catalysed reaction. (a) Representative HPLC showing complete separation of tRNA 1 from tRNA 5. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 175 → 180 mM NaCl over 1 → 20 min. (b) Representative HPLC showing partial separation of tRNA 5 from tRNA 6, and calculated peaks for each tRNA. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 160 → 165 mM NaCl over 1 → 30 min. (c) Michaelis–Menten plot for the FAD-dependent cmnm5s2U → nm5s2U demodification. (d) Michaelis–Menten plot for the SAM-dependent nm5s2U → mnm5s2U methylation. Rates in (c) and (d) represent the amount of substrate formed in a 40 µl reaction per minute per milligram enzyme.
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Figure 3: HPLCs and Michaelis–Menten plots for each MnmC catalysed reaction. (a) Representative HPLC showing complete separation of tRNA 1 from tRNA 5. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 175 → 180 mM NaCl over 1 → 20 min. (b) Representative HPLC showing partial separation of tRNA 5 from tRNA 6, and calculated peaks for each tRNA. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 160 → 165 mM NaCl over 1 → 30 min. (c) Michaelis–Menten plot for the FAD-dependent cmnm5s2U → nm5s2U demodification. (d) Michaelis–Menten plot for the SAM-dependent nm5s2U → mnm5s2U methylation. Rates in (c) and (d) represent the amount of substrate formed in a 40 µl reaction per minute per milligram enzyme.

Mentions: Anion-exchange HPLC of the cmnm5s2U- mnm5s2U- and nm5s2U-containing tRNAs showed that each elutes at a different retention time (Figure 3a and b), allowing an HPLC-based assay to be used to measure both activities of the enzyme. As full separation of tRNA 5 and 6 was not obtained, a computational peak deconvolution was employed to calculate the area of each peak (Figure 3b). Preliminary studies confirmed the linear response of the areas of the product and substrate peaks, and the approximate saturating concentration of the SAM cofactor (∼100 µM). Subsequently, steady-state kinetic assays for each reaction step were carried out. The conditions for each assay were chosen to be similar in order to compare kinetic constants, and were based on conditions known to optimize enzyme activity (20) with the addition of MgCl2 to stabilize tRNA (26). Rate versus substrate concentration curves are shown in Figure 3c and d. Fitting a rectangular hyperbola to these curves gives the Michaelis–Menten constants shown in Table 1. The Km for the second reaction is substantially lower (∼9-fold) than that of first reaction, and the kcat constant is not significantly different. This shows that the enzyme binds the second substrate (tRNA 5) tighter than the first (tRNA 1). This clearly will result in a higher rate of reaction for the second step, or similar rates at very high substrate concentrations where the kcat constant becomes dominant. The kcat/Km value, a general indicator of activity, is correspondingly eight times higher for the second reaction step, showing substantially higher activity at intermediate substrate concentrations.Figure 3.


Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U.

Pearson D, Carell T - Nucleic Acids Res. (2011)

HPLCs and Michaelis–Menten plots for each MnmC catalysed reaction. (a) Representative HPLC showing complete separation of tRNA 1 from tRNA 5. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 175 → 180 mM NaCl over 1 → 20 min. (b) Representative HPLC showing partial separation of tRNA 5 from tRNA 6, and calculated peaks for each tRNA. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 160 → 165 mM NaCl over 1 → 30 min. (c) Michaelis–Menten plot for the FAD-dependent cmnm5s2U → nm5s2U demodification. (d) Michaelis–Menten plot for the SAM-dependent nm5s2U → mnm5s2U methylation. Rates in (c) and (d) represent the amount of substrate formed in a 40 µl reaction per minute per milligram enzyme.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: HPLCs and Michaelis–Menten plots for each MnmC catalysed reaction. (a) Representative HPLC showing complete separation of tRNA 1 from tRNA 5. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 175 → 180 mM NaCl over 1 → 20 min. (b) Representative HPLC showing partial separation of tRNA 5 from tRNA 6, and calculated peaks for each tRNA. HPLC gradient: 100 mM Tris, 50 mM MgCl2, 160 → 165 mM NaCl over 1 → 30 min. (c) Michaelis–Menten plot for the FAD-dependent cmnm5s2U → nm5s2U demodification. (d) Michaelis–Menten plot for the SAM-dependent nm5s2U → mnm5s2U methylation. Rates in (c) and (d) represent the amount of substrate formed in a 40 µl reaction per minute per milligram enzyme.
Mentions: Anion-exchange HPLC of the cmnm5s2U- mnm5s2U- and nm5s2U-containing tRNAs showed that each elutes at a different retention time (Figure 3a and b), allowing an HPLC-based assay to be used to measure both activities of the enzyme. As full separation of tRNA 5 and 6 was not obtained, a computational peak deconvolution was employed to calculate the area of each peak (Figure 3b). Preliminary studies confirmed the linear response of the areas of the product and substrate peaks, and the approximate saturating concentration of the SAM cofactor (∼100 µM). Subsequently, steady-state kinetic assays for each reaction step were carried out. The conditions for each assay were chosen to be similar in order to compare kinetic constants, and were based on conditions known to optimize enzyme activity (20) with the addition of MgCl2 to stabilize tRNA (26). Rate versus substrate concentration curves are shown in Figure 3c and d. Fitting a rectangular hyperbola to these curves gives the Michaelis–Menten constants shown in Table 1. The Km for the second reaction is substantially lower (∼9-fold) than that of first reaction, and the kcat constant is not significantly different. This shows that the enzyme binds the second substrate (tRNA 5) tighter than the first (tRNA 1). This clearly will result in a higher rate of reaction for the second step, or similar rates at very high substrate concentrations where the kcat constant becomes dominant. The kcat/Km value, a general indicator of activity, is correspondingly eight times higher for the second reaction step, showing substantially higher activity at intermediate substrate concentrations.Figure 3.

Bottom Line: Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process.These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations.This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

ABSTRACT
Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.

Show MeSH
Related in: MedlinePlus