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Ribonuclease A

section IX).182,183 One appropriate application of assays using RNA polymers is for the detection of ribonucleolytic activity in a complex mixture. For example, the release of methylene blue from yeast RNA provides a sensitive assay at 688 nm, a wave- length of light not absorbed by most biomolecules. 184

Alternatively, zymogram assays as 1 pg (0.1 fmol) of RNase A. In

can detect as little a zymogram assay,

a

polymeric

substrate

is

incorporated

into

a

gel,

and

cleavage

is

visualized

by

staining

for

intact

polymers

after electrophoresis185-187 or isoelectric A zymogram blot is also effective. 189

focusing.

188

Answering questions about enzymatic catalysis with chemical rigor requires the use of well-defined substrates. Homopolymeric substrates such as poly- (U) and poly(C) are now readily available. Further, the advent of phosphoramidite chemistry has enabled the facile synthesis of any di-, tri-, or tetranucleotide substrate. (For an example, see ref 190.) Uridylyl- (3f5)adenosine (UpA) and cytidylyl(3f5)adenosine (CpA), which have well-defined extinction coeffi- cients,191 have become the most often used oligo- nucleotide substrates. Because RNase A does not catalyze DNA cleavage, the synthesis of RNA/DNA chimeras extends further the horizon of possible analyses. 178,192

A new fluorogenic substrate provides the basis for an extremely sensitive assay for RNase A. 5[-O-[4- [(2,4-Dinitrophenyl)amino]butyl]phosphoryl]uridylyl- (3f5)2-deoxyadenosine 3-[N-[(2-aminobenzoyl)- amino]prop-3-yl] phosphate (DUPAAA; 1) consists of a fluorophore (o-aminobenzoic acid) linked via Ud(pA) to a quencher (2,4-dinitroaniline).193 Cleav- age of the phosphodiester bond in the Ud(pA) linker results in a 60-fold increase in fluorescence, enabling the detection of a 50 fM concentration of RNase A.

1

New chromogenic substrates facilitate assays of RNase A. Uridine 3-(5-bromo-4-chloroindol-3-yl) phosphate (U-3-BCIP; 2) is a substrate for RNase

A.194,195

The

5-bromo-4-chloroindol-3-ol

product

dimer-

izes

rapidly

in

air

to

form

a

blue

pigment.

This

Chemical Reviews, 1998, Vol. 98, No. 3 1051

substrate is analogous to (5-bromo-4-chloroindol-3- yl)galactose (X-gal), a common substrate for -galac- tosidase. Other chromogenic substrates rely on the production of yellow phenolates from the cleavage of uridine 3-aryl phosphates. 37,154,196

VII. Inhibitors

The most potent inhibitor of RNase A, appropri- ately called “ribonuclease inhibitor” (RI), is a 50-kDa protein that constitutes e 0.01% of the protein in the cytosol of mammalian cells.18,197 RI presumably protects cytosolic RNA against the invasion of pan- creatic ribonucleases. The value of Kd for the RIRNase A complex has been measured to be 4.4 × 10-14 M198 and 6.7 × 10-14 M.199 The crystalline structures of RI200 and the RIRNase A complex201,202 disclose that this tight association is due largely to hydrogen bonds and Coulombic interactions. The ability to evade RI appears to be a key attribute of

those homologues section XII.) RI views.203,204

of RNase A that are cytotoxic. (See has been the object of recent re-

Small-molecule inhibitors of RNase A are also known. Nucleosides form complexes with oxovana- dium(IV) and vanadium(V) ions. At least one of these complexes with vanadium(V), uridine 2,3-cyclic vanadate (U>v), is a potent inhibitor of RNase A. Uridine-vanadate complexes have been reported to inhibit RNase A with an apparent Ki near 10 µM.205 In a detailed study, the value of Ki for the U>v species alone has been determined to be near 0.5 µM.206

U>v was conceived as a transition-state analogue for the hydrolysis reaction of RNase A.207 The vanadium in U>v does indeed have a nearly trigonal bipyramidal geometry when bound in the active site of RNase A.164,167 Nevertheless, both theoretical and experimental157 approaches reveal that U>v more closely resembles the ground-state rather than the transition state of the RNase AU>p complex. 208

The most potent noncovalent small-molecule in- hibitors of RNase A are now 5-diphosphoadenosine 3-phosphate (3) and 5-diphosphoadenosine 2-phos- phate.209 The value of Kd for the RNase A3 complex is 0.24 µM, and that for the RNase A5-diphospho- adenosine 2-phosphate complex is 0.52 µM. The structures of crystalline complexes reveal that the bound inhibitors occupy the P1 and B2 subsites.210

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