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Titolo:
RICIN A-CHAIN - KINETICS, MECHANISM, AND RNA STEM-LOOP INHIBITORS
Autore:
CHEN XY; LINK TM; SCHRAMM VL;
Indirizzi:
YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM,1300 MORRIS PK AVEBRONX NY 10461 YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM BRONX NY 10461
Titolo Testata:
Biochemistry
fascicolo: 33, volume: 37, anno: 1998,
pagine: 11605 - 11613
SICI:
0006-2960(1998)37:33<11605:RA-KMA>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
TRANSITION-STATE INHIBITORS; ELECTROSTATIC POTENTIAL SURFACE; RIBOSOME-INACTIVATING PROTEINS; ADENOSINE GLYCOSIDASE ACTIVITY; AMP NUCLEOSIDASE; FORMYCIN 5'-PHOSPHATE; IDENTITY ELEMENTS; SUBSTRATE; SITE; DNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
50
Recensione:
Indirizzi per estratti:
Citazione:
X.Y. Chen et al., "RICIN A-CHAIN - KINETICS, MECHANISM, AND RNA STEM-LOOP INHIBITORS", Biochemistry, 37(33), 1998, pp. 11605-11613

Abstract

Ricin A-chain (RTA) catalyzes the depurination of a single adenine atposition 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 10(5) M-1 s(-1) under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower k(cat) values, but allhave K-m values of similar to 5 mu M. Both the 10- and 18-base substrates have k(cat)/K-m near 10(4) 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 of0.1 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 mu M), 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 mu M) is weaker than substrate which indicates that the altered pK(a) at this position is not an important feature of transitionstate recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (K-i = 0.18 mu M). 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.

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Documento generato il 18/09/20 alle ore 17:15:09