Highly Precise Measurement of Kinetic Isotope Effects Using 1H-Detected 2D [13C,1H]-HSQC NMR Spectroscopy

Manning, Kathryn A.; Sathyamoorthy, Bharathwaj; Eletsky, Alexander; Szyperski, Thomas; Murkin, Andrew S.

doi:10.1021/ja310353c

Cited by 35 publications

(49 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Whereas several previously established methods show capability for precise measurement of KIEs, MALDI-TOF MS was implemented here because of the generality and convenience of this method to probe protein posttranslational modifications with isotopically labeled cofactors (2,3,15,21,47). Unmodified H4K20 peptide displays a dominant characteristic set of six mass multiplets, which maintain fixed ratios of individual peak areas and arise from natural isotope abundance of 13 3A).…”

Section: Quantification Of Isotopic Ratios By Deconvoluted Maldi-tof Msmentioning

confidence: 99%

“…The isotopic ratios of prereacted substrates vs. bound substrates or depleted substrates (or unconsumed substrates) are then quantified for the calculation of BIEs or KIEs, respectively. Several approaches that can precisely determine isotopic ratios of two mixed isomers are remote radioactive labeling, 1D/2D NMR spectroscopy, and isotope ratio MS (16,(21)(22)(23).Protein lysine methyltransferases (PKMTs) belong to a subfamily of posttranslational modifying enzymes (24,25). PKMTs catalyze the methylation of histone and nonhistone protein substrates at targeted lysine residues (26-28).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

show abstract

Section: Quantification Of Isotopic Ratios By Deconvoluted Maldi-tof Msmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The accumulated evidence favored the retro-aldol/ aldol sequence [9][10][11]. The mechanism was finally elucidated via determining the kinetic isotope effects by using 2 H-or 13 C-labeled DXP as substrates [12][13][14]. The a-ketol rearrangement mechanism was definitively ruled out.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic insights into 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway

Tian

Sun

et al. 2013

The FEBS Journal

View full text Add to dashboard Cite

O isotope exchange experiments and determination of the kinetic parameters of the reaction. The results support a C3-C4 substrate binding mode in which DXP chelates a DXR-bound divalent cation via its hydroxyl groups at C3 and C4. Based on this binding mode and the early results, a catalytic cycle for the conversion of DXP to 2-methyl-D-erythritol 4-phosphate mediated by DXR including a pseudo-single molecule transition state of the retro-aldol intermediates is proposed. Taking into account the binding mode of DXP and the catalytic cycle of DXR, the mechanistic insights of DXR are disclosed and the current discrepancies concerning the catalysis of this enzyme are interpreted within the accepted retro-aldol/aldol sequence.

show abstract

“…Complementary to the deuterium KIE determinations, 13 C KIEs were determined by Manning et al [74] for M. tuberculosis DXR using a method based on 2D [ 13 C, 1 H]-HSQC NMR. This highly precise technique employs an internal competition for measurement of KIEs, in which light ( 12 C) and heavy ( 13 C) substrates are reacted simultaneously in the same mixture with the enzyme.…”

Section: Kinetic Isotope Effects For Isomerizationmentioning

confidence: 99%

Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Murkin

Manning

Kholodar

2014

Bioorganic Chemistry

Self Cite

View full text Add to dashboard Cite

Highly Precise Measurement of Kinetic Isotope Effects Using ¹H-Detected 2D [¹³C,¹H]-HSQC NMR Spectroscopy

Cited by 35 publications

References 30 publications

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Mechanistic insights into 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway

Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Contact Info

Product

Resources

About