Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

Yong Zhang, Vern L. Schramm

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large R-secondary 1'-3H kcat/Km kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition- state effects from the k cat/Km KIEs. The BIEs for interactions of [1'-3H]orotidine 50-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp3 bond hybridization of C1' toward the sp2 geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-3H]OMP kcat/K m KIEs are approximately 1.20, half of the intrinsic k cat/Km KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-3H]orotidine BIEs. Thus, OPRTs are unable to initiate groundstate destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the kcat/Km KIEs for [1'-3H]orotidine are also approximately 1.20. The C1' distortion forOMPhappens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.

Original languageEnglish (US)
Pages (from-to)4813-4818
Number of pages6
JournalBiochemistry
Volume50
Issue number21
DOIs
StatePublished - May 31 2011

Fingerprint

Orotate Phosphoribosyltransferase
Isotopes
Ground state
Kinetics
Catalytic Domain
Cats
orotidine
Substrates
Pyrimidines
Plasmodium falciparum

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects. / Zhang, Yong; Schramm, Vern L.

In: Biochemistry, Vol. 50, No. 21, 31.05.2011, p. 4813-4818.

Research output: Contribution to journalArticle

@article{72d52f384a3149338d19209d4d7d7b73,
title = "Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects",
abstract = "Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large R-secondary 1'-3H kcat/Km kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition- state effects from the k cat/Km KIEs. The BIEs for interactions of [1'-3H]orotidine 50-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp3 bond hybridization of C1' toward the sp2 geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-3H]OMP kcat/K m KIEs are approximately 1.20, half of the intrinsic k cat/Km KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-3H]orotidine BIEs. Thus, OPRTs are unable to initiate groundstate destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the kcat/Km KIEs for [1'-3H]orotidine are also approximately 1.20. The C1' distortion forOMPhappens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.",
author = "Yong Zhang and Schramm, {Vern L.}",
year = "2011",
month = "5",
day = "31",
doi = "10.1021/bi200638x",
language = "English (US)",
volume = "50",
pages = "4813--4818",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

AU - Zhang, Yong

AU - Schramm, Vern L.

PY - 2011/5/31

Y1 - 2011/5/31

N2 - Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large R-secondary 1'-3H kcat/Km kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition- state effects from the k cat/Km KIEs. The BIEs for interactions of [1'-3H]orotidine 50-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp3 bond hybridization of C1' toward the sp2 geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-3H]OMP kcat/K m KIEs are approximately 1.20, half of the intrinsic k cat/Km KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-3H]orotidine BIEs. Thus, OPRTs are unable to initiate groundstate destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the kcat/Km KIEs for [1'-3H]orotidine are also approximately 1.20. The C1' distortion forOMPhappens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.

AB - Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large R-secondary 1'-3H kcat/Km kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition- state effects from the k cat/Km KIEs. The BIEs for interactions of [1'-3H]orotidine 50-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp3 bond hybridization of C1' toward the sp2 geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-3H]OMP kcat/K m KIEs are approximately 1.20, half of the intrinsic k cat/Km KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-3H]orotidine BIEs. Thus, OPRTs are unable to initiate groundstate destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the kcat/Km KIEs for [1'-3H]orotidine are also approximately 1.20. The C1' distortion forOMPhappens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.

UR - http://www.scopus.com/inward/record.url?scp=79958118740&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79958118740&partnerID=8YFLogxK

U2 - 10.1021/bi200638x

DO - 10.1021/bi200638x

M3 - Article

VL - 50

SP - 4813

EP - 4818

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -