Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata

Horenstein, Benjamin A.; Parkin, David W.; Estupiñán, B; Schramm, Vern L.

doi:10.1021/bi00108a026

Cited by 156 publications

(270 citation statements)

References 18 publications

(30 reference statements)

Supporting

Mentioning

242

Contrasting

Order By: Relevance

“…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 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

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

mentioning

confidence: 99%

mentioning

confidence: 99%

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 IU-NHs were scrutinized by V. Schramm and co-workers (11,12). For this subclass of enzymes an S n 1 reaction mechanism with a ribosyl oxocarbenium-like transition state was established by kinetic isotope effect studies (11,12).…”

mentioning

confidence: 99%

Enzyme-Substrate Interactions in the Purine-specific Nucleoside Hydrolase from Trypanosoma vivax

Versées

Decanniere

Holsbeke

et al. 2002

Journal of Biological Chemistry

131

View full text Add to dashboard Cite

Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosineguanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (1). Here we report the 2.0-Å crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-Å crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2-OH, 3-OH, and 5-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k cat /K m , respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp 83 and Trp 260. The pH profiles of k cat and k cat /K m indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation.

show abstract

“…For example, nucleosidases are responsible for hydrolyzing nucleosidic bonds. The transition state of inosine nucleosidase is characterized by formation of an oxocarbenium ion character that develops concurrently with the flattening of the ribofuranosyl ring [53]. In this case, the non-bonding electrons of the incoming water are proposed to stabilize the positive charge [53].…”

Section: Trna-guanine Transgylcosylasementioning

confidence: 99%

“…The transition state of inosine nucleosidase is characterized by formation of an oxocarbenium ion character that develops concurrently with the flattening of the ribofuranosyl ring [53]. In this case, the non-bonding electrons of the incoming water are proposed to stabilize the positive charge [53]. Likewise, the DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA.…”

Section: Trna-guanine Transgylcosylasementioning

confidence: 99%

Transglycosylation: A mechanism for RNA modification (and editing?)

Garcia

Kittendorf

2005

Bioorganic Chemistry

View full text Add to dashboard Cite

The vast majority of the ca. 100 chemically distinct modified nucleosides in RNA appear to arise via the chemical transformation of a genetically encoded nucleoside. Two notable exceptions are queuosine and pseudouridine, which are incorporated into tRNA via transglycosylation. Transglycosylation is an extremely efficient process for incorporating highly modified bases such as queuine into RNA. Transglycosylation is also a requisite process for "isomerizing" an Nnucleoside into a C-nucleoside as is the case for pseudouridine formation. Finally, transglycosylation is an attractive possibility for certain RNA editing events (e.g., pyrimidine to purine conversions) that cannot occur via the known, more straightforward enzymatic reactions (e.g., deaminations). This review discusses what is known about the mechanisms of transglycosylation for the queuine and pseudouridine RNA modifications and will speculate about a potential role for transglycosylation in certain RNA editing events.

show abstract

Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata

Cited by 156 publications

References 18 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

Enzyme-Substrate Interactions in the Purine-specific Nucleoside Hydrolase from Trypanosoma vivax

Transglycosylation: A mechanism for RNA modification (and editing?)

Contact Info

Product

Resources

About