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1052 Chemical Reviews, 1998, Vol. 98, No. 3

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

212 213

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-O5bond 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.

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