Ricin A-chain

Kinetics, mechanism, and RNA stem-loop inhibitors

Xiang Yang Chen, Todd M. Link, Vern L. Schramm

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a k(cat)/K(m) of 4.5 x 105 M-1 s- 1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K(m) values of ~5 μM. Both the 10- and 18-base substrates have k(cat)/K(m) near 104 M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem- loop DNA (10 bases) of the same sequence is also a substrate with a k(cat)/K(m) of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O- riboside at the depurination site is not a substrate, but binds tightly to the enzyme (K(i) = 0.34 μM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pK(a) of the adenine analogue at N7. Binding of this analogue (K(i) = 9.4 μM) is weaker than substrate which indicates that the altered pK(a) at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (K(i) = 0.18 μM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.

Original languageEnglish (US)
Pages (from-to)11605-11613
Number of pages9
JournalBiochemistry
Volume37
Issue number33
DOIs
StatePublished - Aug 18 1998

Fingerprint

Ricin
RNA
Kinetics
Substrates
Rapid thermal annealing
Adenine
Catalysis
Hydroxyl Radical
Ionization
Chemical activation
Protonation
DNA
Enzymes
Ribosomes
Kinetic parameters
Thermodynamic properties
Nucleotides
Stabilization
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ricin A-chain : Kinetics, mechanism, and RNA stem-loop inhibitors. / Chen, Xiang Yang; Link, Todd M.; Schramm, Vern L.

In: Biochemistry, Vol. 37, No. 33, 18.08.1998, p. 11605-11613.

Research output: Contribution to journalArticle

Chen, Xiang Yang ; Link, Todd M. ; Schramm, Vern L. / Ricin A-chain : Kinetics, mechanism, and RNA stem-loop inhibitors. In: Biochemistry. 1998 ; Vol. 37, No. 33. pp. 11605-11613.
@article{5d34672efe54475da24d85af2b3a3d09,
title = "Ricin A-chain: Kinetics, mechanism, and RNA stem-loop inhibitors",
abstract = "Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a k(cat)/K(m) of 4.5 x 105 M-1 s- 1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K(m) values of ~5 μM. Both the 10- and 18-base substrates have k(cat)/K(m) near 104 M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem- loop DNA (10 bases) of the same sequence is also a substrate with a k(cat)/K(m) of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O- riboside at the depurination site is not a substrate, but binds tightly to the enzyme (K(i) = 0.34 μM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pK(a) of the adenine analogue at N7. Binding of this analogue (K(i) = 9.4 μM) is weaker than substrate which indicates that the altered pK(a) at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (K(i) = 0.18 μM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.",
author = "Chen, {Xiang Yang} and Link, {Todd M.} and Schramm, {Vern L.}",
year = "1998",
month = "8",
day = "18",
doi = "10.1021/bi980990p",
language = "English (US)",
volume = "37",
pages = "11605--11613",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "33",

}

TY - JOUR

T1 - Ricin A-chain

T2 - Kinetics, mechanism, and RNA stem-loop inhibitors

AU - Chen, Xiang Yang

AU - Link, Todd M.

AU - Schramm, Vern L.

PY - 1998/8/18

Y1 - 1998/8/18

N2 - Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a k(cat)/K(m) of 4.5 x 105 M-1 s- 1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K(m) values of ~5 μM. Both the 10- and 18-base substrates have k(cat)/K(m) near 104 M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem- loop DNA (10 bases) of the same sequence is also a substrate with a k(cat)/K(m) of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O- riboside at the depurination site is not a substrate, but binds tightly to the enzyme (K(i) = 0.34 μM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pK(a) of the adenine analogue at N7. Binding of this analogue (K(i) = 9.4 μM) is weaker than substrate which indicates that the altered pK(a) at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (K(i) = 0.18 μM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.

AB - Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a k(cat)/K(m) of 4.5 x 105 M-1 s- 1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but all have K(m) values of ~5 μM. Both the 10- and 18-base substrates have k(cat)/K(m) near 104 M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem- loop DNA (10 bases) of the same sequence is also a substrate with a k(cat)/K(m) of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O- riboside at the depurination site is not a substrate, but binds tightly to the enzyme (K(i) = 0.34 μM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pK(a) of the adenine analogue at N7. Binding of this analogue (K(i) = 9.4 μM) is weaker than substrate which indicates that the altered pK(a) at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (K(i) = 0.18 μM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.

UR - http://www.scopus.com/inward/record.url?scp=0032544216&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032544216&partnerID=8YFLogxK

U2 - 10.1021/bi980990p

DO - 10.1021/bi980990p

M3 - Article

VL - 37

SP - 11605

EP - 11613

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 33

ER -