Transition state structure of 5′-methylthioadenosine/S- adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues

Vipender Singh, Jeffrey E. Lee, Sara Núñez, P. Lynne Howell, Vern L. Schramm

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5′-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1′-3H, 1′-14C, 2′-3H, 4′-3H, 5′-3H, 9-15N, and Me-3H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1′-3H and small 1′-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 Å from the anomeric carbon. The small β-secondary 2′-3H KIE of 1.044 corresponds to a modest 3′-endo conformation for ribose and a H1′-C1′- C2′-H2′ dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4′-3H KIE is due to hyperconjugation between the lone pair (np) of O3′ and the antibonding (σ*) orbital of the C4′-H4′ group, and the methyl-3H3 KIE is due to hyperconjugation between the np of sulfur and the σ* of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.

Original languageEnglish (US)
Pages (from-to)11647-11659
Number of pages13
JournalBiochemistry
Volume44
Issue number35
DOIs
StatePublished - Sep 6 2005

Fingerprint

adenosylhomocysteine nucleosidase
Isotopes
Escherichia coli
Kinetics
Pemetrexed
Salvaging
Quorum Sensing
Nucleophiles
Methylation
Ribose
Drug Design
Polyamines
Adenine
Dihedral angle
Static Electricity
Catalysis
Sulfur
Metabolism

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition state structure of 5′-methylthioadenosine/S- adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues. / Singh, Vipender; Lee, Jeffrey E.; Núñez, Sara; Howell, P. Lynne; Schramm, Vern L.

In: Biochemistry, Vol. 44, No. 35, 06.09.2005, p. 11647-11659.

Research output: Contribution to journalArticle

@article{b730ef8e479e4296b29ec9d721076a03,
title = "Transition state structure of 5′-methylthioadenosine/S- adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues",
abstract = "Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5′-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1′-3H, 1′-14C, 2′-3H, 4′-3H, 5′-3H, 9-15N, and Me-3H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1′-3H and small 1′-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 {\AA} from the anomeric carbon. The small β-secondary 2′-3H KIE of 1.044 corresponds to a modest 3′-endo conformation for ribose and a H1′-C1′- C2′-H2′ dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4′-3H KIE is due to hyperconjugation between the lone pair (np) of O3′ and the antibonding (σ*) orbital of the C4′-H4′ group, and the methyl-3H3 KIE is due to hyperconjugation between the np of sulfur and the σ* of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.",
author = "Vipender Singh and Lee, {Jeffrey E.} and Sara N{\'u}{\~n}ez and Howell, {P. Lynne} and Schramm, {Vern L.}",
year = "2005",
month = "9",
day = "6",
doi = "10.1021/bi050863a",
language = "English (US)",
volume = "44",
pages = "11647--11659",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "35",

}

TY - JOUR

T1 - Transition state structure of 5′-methylthioadenosine/S- adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues

AU - Singh, Vipender

AU - Lee, Jeffrey E.

AU - Núñez, Sara

AU - Howell, P. Lynne

AU - Schramm, Vern L.

PY - 2005/9/6

Y1 - 2005/9/6

N2 - Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5′-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1′-3H, 1′-14C, 2′-3H, 4′-3H, 5′-3H, 9-15N, and Me-3H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1′-3H and small 1′-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 Å from the anomeric carbon. The small β-secondary 2′-3H KIE of 1.044 corresponds to a modest 3′-endo conformation for ribose and a H1′-C1′- C2′-H2′ dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4′-3H KIE is due to hyperconjugation between the lone pair (np) of O3′ and the antibonding (σ*) orbital of the C4′-H4′ group, and the methyl-3H3 KIE is due to hyperconjugation between the np of sulfur and the σ* of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.

AB - Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5′-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1′-3H, 1′-14C, 2′-3H, 4′-3H, 5′-3H, 9-15N, and Me-3H3 were 1.160 ± 0.004, 1.004 ± 0.003, 1.044 ± 0.004, 1.015 ± 0.002, 1.010 ± 0.002, 1.018 ± 0.006, and 1.051 ± 0.002, respectively. The large 1′-3H and small 1′-14C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (DN*AN) (SN1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 Å from the anomeric carbon. The small β-secondary 2′-3H KIE of 1.044 corresponds to a modest 3′-endo conformation for ribose and a H1′-C1′- C2′-H2′ dihedral angle of 53° at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4′-3H KIE is due to hyperconjugation between the lone pair (np) of O3′ and the antibonding (σ*) orbital of the C4′-H4′ group, and the methyl-3H3 KIE is due to hyperconjugation between the np of sulfur and the σ* of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.

UR - http://www.scopus.com/inward/record.url?scp=23944446462&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=23944446462&partnerID=8YFLogxK

U2 - 10.1021/bi050863a

DO - 10.1021/bi050863a

M3 - Article

C2 - 16128565

AN - SCOPUS:23944446462

VL - 44

SP - 11647

EP - 11659

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 35

ER -