Isotope and Elemental Effects Indicate a Rate-Limiting Methyl Transfer as the Initial Step in the Reaction Catalyzed by <i>Escherichia</i> <i>coli</i> Cyclopropane Fatty Acid Synthase

Iwig, David F.; Grippe, Anthony T.; McIntyre, Timothy A.; Booker, Squire J.

doi:10.1021/bi048692h

Cited by 63 publications

(69 citation statements)

References 62 publications

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“…Along the reaction path, the stiffness of the methyl group is further enhanced from SET8-bound SAM to the TS as reflected by a larger inverse CD3 k of 0.90. Several previous studies have reported inverse CD 3 KIEs for SAM-dependent methyltransferases and proposed that the S-N motions tighten the vibrational modes of the sulfonium-methyl moiety at the S N 2 TS (18,55,56). Our KIE measurements together with QM modeling suggest that SET8-catalyzed methylation adopts an early, asymmetrical S N 2 mechanism with more axial compression between the methyl donor and acceptor at the TS relative to the unbound and SET8-bound ground states of SAM (Fig.…”

Section: Computational Modeling Of Transition State Of Set8-catalyzedsupporting

confidence: 58%

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.

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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...

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Section: Computational Modeling Of Transition State Of Set8-catalyzedsupporting

confidence: 58%

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.

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“…3,57 S -Adenosylhomocysteine nucleosidase (SAHN) and SAM were prepared as described previously. 58,59 [8,8,8- 2 H 3 ]-Octanoyl-H protein (OHP) was synthesized using Ec LplA as described previously. 3,32 …”

Section: Methodsmentioning

confidence: 99%

Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase

et al. 2014

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Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA•). In the LS reaction, two equivalents of 5′-dA• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]0 clusters.

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“…This enhanced reactivity of AdoSeMet (33) implies that its methyl group is more reactive towards nucleophiles, which is consistent with an enhanced reactivity of trimethylselenonium hydroxide compared to trimethylsulfonium hydroxide [116]. These chemical differences between the three cofactors were used to study the enzymatic mechanism of CFA synthase acting on an isolated double bond [117]. The turnover number (TON) with AdoSeMet (33) is increased, whereas that with AdoTeMet (34) is decreased relative to AdoMet (1).…”

Section: Selenium and Tellurium Analogues Of Adometmentioning

confidence: 59%

S‐Adenosyl‐L‐Methionine and Related Compounds

Dalhoff¹,

Weinhold

2008

Modified Nucleosides

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The sulfonium compound S-adenosyl-L-methionine (AdoMet, also known as SAM or SAMe, 1) is one of the most versatile biomolecules in nature and one of the most widely used enzyme substrates, second perhaps only to adenosine triphosphate (ATP) (Schemes 9.1 and 9.2). AdoMet (1) may be found in all phyla of life, and is one of the molecules crucial for the existence of living organisms. First identified by Cantoni in 1953 [1, 2] as the active methyl donor formed from L-methionine and ATP, AdoMet (1) proved to be not only the major methyl donor in nature but also to be involved in many other metabolic reactions [3,4]. The versatility of the cofactor is based on the reactivity of the pivotal sulfonium center, which activates the adjacent carbon atoms for nucleophilic attack, deprotonation, and radical formation. This leads to a unique cofactor in which all constituents are used in biochemical reactions. The group transfer reactions result in the release of one of three different thioether products which are more stable than the sulfonium compound, and thereby providing the driving force for these enzyme-catalyzed reactions. These highly preferable thermodynamics of AdoMet-dependent transfer reactions result in a strong preference for this cofactor compared, for example, to other methyl donors such as N5-methyltetrahydrofolate. Due to the electron lone pair in addition to the three different substituents, the sulfonium group is chiral, and the naturally occurring AdoMet (1) is S-configured at sulfur [5].Many other naturally occurring alkylated thioadenosines are derived from AdoMet (1) such as S-adenosyl-L-homocysteine (AdoHcy, also called SAH, 2) and 5 0 -deoxy-5 0 -methylthioadenosine (MTA, 3) (Scheme 9.3), although these compounds and their related 5 0 -thioethers are beyond the scope of this chapter. AdoMet (1) and its derivatives are involved in key metabolic reactions, cell cycle regulation and the storage of epigenetic information, which makes them not only highly interesting for studying biological pathways but also as in-vitro or in-vivo biochemical tools and candidates for diagnostics and therapeutics.

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Isotope and Elemental Effects Indicate a Rate-Limiting Methyl Transfer as the Initial Step in the Reaction Catalyzed by Escherichia coli Cyclopropane Fatty Acid Synthase

Cited by 63 publications

References 62 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

Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase

S‐Adenosyl‐L‐Methionine and Related Compounds

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