Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases

Anthony A. Sauve, Sean M. Cahill, Stephan G. Zech, Luiz A. Basso, Andrzej Lewandowicz, Diogenes S. Santos, Charles Grubmeyer, Gary B. Evans, Richard H. Furneaux, Peter C. Tyler, Ann McDermott, Mark E. Girvin, Vern L. Schramm

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated α-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1′-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5′-PO4 (ImmHP) resemble the transition state charge when protonated at 4′-N and bind tightly to these enzymes with Kd values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4′-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP•ImmH•PO4 and HGPRTase•ImmHP•MgPPi indicate protonation at N-4′ for the tightly bound inhibitors. The 1′-13C and 1′-1H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1′-C toward sp2 geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4′-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

Original languageEnglish (US)
Pages (from-to)5694-5705
Number of pages12
JournalBiochemistry
Volume42
Issue number19
DOIs
StatePublished - May 20 2003

Fingerprint

Hypoxanthine Phosphoribosyltransferase
Catalytic Domain
Purine-Nucleoside Phosphorylase
Protonation
Substrates
Stabilization
Ions
Purine Nucleosides
Purine Nucleotides
Nucleophiles
Ribose
Geometry
Guanosine
Enzymes
Binding energy
Static Electricity
Ionization
Anions
Electrostatics
Phosphates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases. / Sauve, Anthony A.; Cahill, Sean M.; Zech, Stephan G.; Basso, Luiz A.; Lewandowicz, Andrzej; Santos, Diogenes S.; Grubmeyer, Charles; Evans, Gary B.; Furneaux, Richard H.; Tyler, Peter C.; McDermott, Ann; Girvin, Mark E.; Schramm, Vern L.

In: Biochemistry, Vol. 42, No. 19, 20.05.2003, p. 5694-5705.

Research output: Contribution to journalArticle

Sauve, AA, Cahill, SM, Zech, SG, Basso, LA, Lewandowicz, A, Santos, DS, Grubmeyer, C, Evans, GB, Furneaux, RH, Tyler, PC, McDermott, A, Girvin, ME & Schramm, VL 2003, 'Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases', Biochemistry, vol. 42, no. 19, pp. 5694-5705. https://doi.org/10.1021/bi034003a
Sauve, Anthony A. ; Cahill, Sean M. ; Zech, Stephan G. ; Basso, Luiz A. ; Lewandowicz, Andrzej ; Santos, Diogenes S. ; Grubmeyer, Charles ; Evans, Gary B. ; Furneaux, Richard H. ; Tyler, Peter C. ; McDermott, Ann ; Girvin, Mark E. ; Schramm, Vern L. / Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases. In: Biochemistry. 2003 ; Vol. 42, No. 19. pp. 5694-5705.
@article{4f5944eec54643d4ad65be04c7946247,
title = "Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases",
abstract = "Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated α-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1′-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5′-PO4 (ImmHP) resemble the transition state charge when protonated at 4′-N and bind tightly to these enzymes with Kd values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4′-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP•ImmH•PO4 and HGPRTase•ImmHP•MgPPi indicate protonation at N-4′ for the tightly bound inhibitors. The 1′-13C and 1′-1H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1′-C toward sp2 geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4′-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.",
author = "Sauve, {Anthony A.} and Cahill, {Sean M.} and Zech, {Stephan G.} and Basso, {Luiz A.} and Andrzej Lewandowicz and Santos, {Diogenes S.} and Charles Grubmeyer and Evans, {Gary B.} and Furneaux, {Richard H.} and Tyler, {Peter C.} and Ann McDermott and Girvin, {Mark E.} and Schramm, {Vern L.}",
year = "2003",
month = "5",
day = "20",
doi = "10.1021/bi034003a",
language = "English (US)",
volume = "42",
pages = "5694--5705",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "19",

}

TY - JOUR

T1 - Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases

AU - Sauve, Anthony A.

AU - Cahill, Sean M.

AU - Zech, Stephan G.

AU - Basso, Luiz A.

AU - Lewandowicz, Andrzej

AU - Santos, Diogenes S.

AU - Grubmeyer, Charles

AU - Evans, Gary B.

AU - Furneaux, Richard H.

AU - Tyler, Peter C.

AU - McDermott, Ann

AU - Girvin, Mark E.

AU - Schramm, Vern L.

PY - 2003/5/20

Y1 - 2003/5/20

N2 - Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated α-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1′-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5′-PO4 (ImmHP) resemble the transition state charge when protonated at 4′-N and bind tightly to these enzymes with Kd values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4′-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP•ImmH•PO4 and HGPRTase•ImmHP•MgPPi indicate protonation at N-4′ for the tightly bound inhibitors. The 1′-13C and 1′-1H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1′-C toward sp2 geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4′-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

AB - Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated α-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1′-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5′-PO4 (ImmHP) resemble the transition state charge when protonated at 4′-N and bind tightly to these enzymes with Kd values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4′-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP•ImmH•PO4 and HGPRTase•ImmHP•MgPPi indicate protonation at N-4′ for the tightly bound inhibitors. The 1′-13C and 1′-1H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1′-C toward sp2 geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4′-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

UR - http://www.scopus.com/inward/record.url?scp=0038290447&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038290447&partnerID=8YFLogxK

U2 - 10.1021/bi034003a

DO - 10.1021/bi034003a

M3 - Article

VL - 42

SP - 5694

EP - 5705

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 19

ER -