Ricin A-chain: kinetics, mechanism, and RNA stem-loop inhibitors.
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 kcat/Km of 4.5 x 10(5) M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower kcat values, but all have Km values of approximately 5 microM. Both the 10- and 18-base substrates have kcat/Km 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 kcat/Km 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 (Ki = 0.34 microM), 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 pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 microM) is weaker than substrate which indicates that the altered pKa 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 (Ki = 0.18 microM). 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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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
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D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D005573:Formycins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D018749:RNA, Plant / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012255:Ribitol / Q000031:analogs & derivatives / Q000494:pharmacology; D012276:Ricin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005573:Formycins; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D018749:RNA, Plant; D012339:RNA, Ribosomal, 28S; C089322:phenyliminoribitol; D012255:Ribitol; C034550:formycin; D012276:Ricin; D009699:N-Glycosyl Hydrolases