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?-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

ABSTRACT: The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or associative with early to late transition states. We have measured ?-secondary kinetic isotope effects operating through four bonds for substitution reactions with dimethylallyl derivatives bearing deuterated methyl groups at the distal (C3) carbon atom in the double bond under dissociative and associative conditions. Computational studies with density functional theory indicate that the magnitudes of the isotope effects correlate with the extent of bond formation between the allylic moiety and the electron-rich acceptor in the transition state for alkylation and provide insights into the structures of the transition states for associative and dissociative alkylation reactions.

SUBMITTER: Choi SR

PROVIDER: S-EPMC4004232 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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δ-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

Choi Seoung-ryoung SR Breugst Martin M Houk Kendall N KN Poulter C Dale CD

The Journal of organic chemistry 20140401 8

The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or assoc ...[more]

PMID: 24665882

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Project description: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 (SN2) 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 MS-based 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 13C KIE of 1.04, an inverse intrinsic ?-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 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 steady-state 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.

Project description:1. The hydrolyses of the p-nitrophenyl esters of N-benzyloxycarbonylglycine, alpha-N-benzyloxycarbonyl-L-lysine and N-methoxycarbonyl-L-phenylalanylglycine catalysed by papain (EC 3.4.22.2) have been studied in solvents having a variable composition of 2H2O and H2O. 2. kcat., which represents deacylation in the papain-catalysed hydrolysis of reactive esters, is some 2.3-fold less in 2H2O compared with H2O. The magnitude of kcat. has been determined as a function of the 2H atom fraction of the solvent. 3. Both linear and non-linear methods of least-square regression analysis have been applied to the data in order to obtain best-fit parameter values for several three-parameter models which express kcat. in terms of the 2H atom fraction of the solvent. These models represent some possible modes of restructuring of the active site protonic configuration consequent upon transition state formation. 4. The results of curve fitting reveal an essentially linear dependence of kcat. upon the 2H atom fraction, and it may therefore be concluded that the isotope effect originates from a single proton which is in the process of transfer in the transition state. 5. It is postulated on the basis of this and other evidence that the mobile proton is transferred from an attacking water molecule to the imidazole side chain of His-159 during tetrahedral intermediate formation. This has the effect of stabilizing the transition state and promoting catalysis. The role of His-159 in deacylation is therefore to provide general base catalysis. 6. Models that involve two or more protons, such as a two-proton relay system analogous to that proposed for the serine proteinases, or a multiproton 'medium' effect, are considered unlikely on the basis of the data reported in this paper. 7. A more detailed examination of possible transition state structures reveals that the only structure compatible with available experimental data and consistent with certain theoretical predictions is one in which the proton translocated in concern with reorganization of the heavy atom framework. In addition, the transition state vibrations of the mobile proton are strongly coupled to those of the heavy atoms. These properties of the transition state are also manifest in the transition state for the deacylation of serine proteinases.

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?-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

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δ-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

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