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Nucleophilic aromatic substitution by poly(A) on 1-fluoro-2,4-dinitrobenzene yields poly[2′-O-(2,4-dini- trophenyl)]poly(adenylic acid) [(DNP-poly(A)].211 DNP- poly(A) of molecular mass 110 kDa and DNP:adenine ratio of 1:1.5 is a reversible competitive inhibitor but not a substrate of RNase A, RNase B, RNase S (see
section values RNase
X), and other ribonucleases.212 The IC50 for inhibition of RNase A, RNase B, and S by DNP-poly(A) have been reported to be
3.20, 0.50, and 0.08 µM, respectively. ing DNP-poly(A) within porous gels
Encapsulat- or attaching
it to acrylic beads212 generates affinity matrixes that effectively remove RNase A from solution. Bound RNase A can be eluted from these matrixes by washing with aqueous solutions of high ionic strength.
Specific affinity labels for RNase A exist. 6-Chlo- ropurine 9--D-ribofuranosyl 5′-monophosphate (4) alkylates the R-amino group of Lys1, presumably after binding to the B3 subsite (Figure 2).150,151 The
structure of the crystalline product of the alkylation of RNase A by 4 is known.152 2′-(3′)-O-Bromoacety- luridine214,215 and its amide analogues 3′-(bromoac- etamido)-3′-deoxythymidine (5), 3′-(bromoacetamido)- 3′-deoxyuridine, 3′-(bromoacetamido)-3′-deoxyarabino- furanosyluracil, 2′-(bromoacetamido)-2′-deoxyuridine,
2′-(bromoacetamido)-2′-deoxyxylofuranosyl- alkylate the side chains of His12 or and uracil216-218
His119. The structures of the crystalline products of the alkylation of RNase A by 5 and by 3′- (bromoacetamido)-3′-deoxyuridine are known. 219
Finally, RNase A has been the object of mecha- nism-based inactivation. The enzyme catalyzes the conversion of uridine 3′-[4-(fluoromethyl)phenyl] phos- phate (6) to a quinone methide, which likely alkylates the side chain of Lys7, Arg10, Gln69, or Glu111 (Figure 2).220 None of these residues are in the active site, and approximately one-third of the catalytic activity remains after alkylation.
VIII. Reaction Mechanism
RNase A catalyzes the cleavage of the P-O5′ bond of RNA. Figure 5 depicts a mechanism of catalysis that is consistent with all known data from work on the enzyme itself.221 Other mechanisms have also been proposed (vide infra).222-225 In the mechanism in Figure 5, the side chain of His12 acts as a base that abstracts a proton from the 2′-oxygen of a substrate molecule, and thereby facilitates its attack on the phosphorus atom. This attack proceeds in- line to displace a nucleoside.226,227 The side chain of His119 acts as an acid that protonates the 5′′-oxygen to facilitate its displacement. Both products are released to solvent. The slow hydrolysis of the nucleoside 2′,3′-cyclic phosphodiester occurs in a separate process (see section IX), and resembles the
Figure 5. (A) Putative mechanism for the transphospho- rylation reaction catalyzed by RNase A and (B) putative mechanism for the hydrolysis reaction catalyzed by RNase A.221 In both mechanisms, “B” is His12 and “A” is His119.