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Ribonuclease A

Three of the enzymic subsites (B1, B2, and B3) interact with the bases of a bound substrate. The B1 subsite appears to bind only pyrimidine bases,74,81 and demonstrates an approximately 30-fold kinetic preference for cytosine-containing versus uracil- containing substrates. In contrast, the B2 and B3 subsites bind all bases, but B2 has a preference for

an adenine base purine base. 143 144,145

and B3 has a preference for a Site-directed mutagenesis has

been used to identify the most important residues in the B1146-148 and B2149 subsites. The existence of the B3 subsite has been inferred from kinetic data144,145

and chemical modification studies.150-152 In crystalline RNase Ad(ApTpApApG) complex,

the the

adenine adenine

base in the B3 subsite base in the B2 subsite.77

stacks

with the

The B3

“subsite”

could

therefore

result

from

π-π

stacking

interactions

that

stabilize

the

enzyme-nucleic

acid

complex

solely

by

preorganization

or

desolvation

of

the

nucleic

acid.

Three other enzymic subsites (P0, P1, and P2) interact with the phosphoryl groups of a bound substrate.141 The enzyme catalyzes the cleavage of the P-O5bond of a phosphoryl group bound in the P1 subsite, which is the active site (Figure 2). Site- directed mutagenesis has been used to identify the

most important residues in the P137,46,153-157

and

P2158,159 subsites. The existence of the P0 subsite has been inferred from kinetic data,160,161 molecular mod- eling,162 and the results of recent site-directed mu- tagenesis experiments. 159

B. Substrate Specificity

RNase A catalyzes the cleavage of the P-O5bond of an RNA strand and the hydrolysis of the P-O2bond of a nucleoside 2,3-cyclic phosphodiester (N>p) on the 3-side of a pyrimidine residue. CpX is cleaved and C>p is hydrolyzed 2-fold faster than are the corresponding uridylyl substrates. (For a review, see ref 16.) Poly(C) is cleaved approximately 20-fold

faster

than

is

poly(U).146,163

RNase

A

will

also

146,163 catalyze the cleavage of 103- to 104-fold less than (U).

poly(A), but at a rate that is that for the cleavage of poly-

The side-chain hydroxyl and main-chain carbonyl

groups of Thr45 mediate the pyrimidine specificity of RNase A by forming hydrogen bonds to a pyrimi- dine base and by excluding sterically a purine base. In the structure of RNase A with uridine 2,3-cyclic vanadate (U>v; see section VII), the Oγ1-N3 distance is 2.7 Å with a Oγ1-H-N3 angle of 147°, and the N - O 2 d i s t a n c e i s 2 . 6 Å w i t h a N - H - O 2 a n g l e o f 1 4 7 ° . 1 6 4 T h e s i d e c h a i n o f P h e 1 2 0 m a k e s v a Waals contact with a pyrimidine base bound in the B1 subsite. The side chain of Ser123 has been assumed to form a hydrogen bond to a uracil bound in the B1 subsite, and to thereby enhance the rate of cleavage after uridine residues.165,166 Such a hydro- gen bond, however, is not evident in the RNase A 146 n d e r

complex with U>v.164,167

Moreover, replacing the

analogous serine in angiogenin, a homologue of RNase A, has no effect on substrate specificity. 168

Site-directed mutagenesis has been used to create variants that cleave efficiently after a purine residue. Enzyme libraries were created in which all 20 amino acid residues replaced Thr45 or Phe120.147 Screening these libraries revealed that replacing Thr45 with a

Chemical Reviews, 1998, Vol. 98, No. 3 1049

Figure 3. (A) Hydrogen bonds formed between a bound cytidine nucleotide and the residues of the B1 subsite of RNase A79 and (B) hydrogen bonds formed between a bound uridine nucleotide and the residues of the B1 subsite of RNase A.164

glycine or alanine residue enables RNase A to cleave poly(A) efficiently.146 The T45G and T45A enzymes have 105- and 103-fold increases, respectively, in poly- (A):poly(C) specificity with little compromise to cata- lytic efficacy. With its diminished substrate speci- ficity, T45G RNase A is more effective than is the wild-type enzyme at degrading heteropolymeric RNA to completion,169 which could be advantageous in ribonuclease protection assays. 170

The interaction between Asp83 and Thr45 also affects the specificity of RNase A. Thermodynamic cycles with the T45G, D83A, and T45G/D83A vari- ants indicate that the side chain of Asp83 has no effect on the kinetics of cleavage after cytidine residues, but does affect significantly the rate of cleavage of poly(U) and hydrolysis of U>p through an interaction that is dependent on the side chain of Thr45 (Figure 3).148 Apparently, the Thr45-Asp83 hydrogen bond increases the ability of RNase A to cleave uridine-containing substrates by the selective stabilization of the transition state for this reaction. These results indicate that like a direct interaction between an enzyme and its substrate, an interaction between two functional groups within an enzyme can contribute to substrate specificity.

No alteration of Phe120 produced an enzyme that catalyzes the efficient cleavage of RNA after purine residues.146 This result is consistent with two struc- tural features of Phe120 that are apparent in the RNase AU>v complex.164,171 First, the aromatic ring of Phe120 appears to interact with a pyrimidine base bound in the B1 subsite. The structural difference between a pyrimidine base and a purine base is largely two-dimensional, in the plane of the π-system. Hence, the side chain of Phe120 does not mediate purine:pyrimidine specificity, but acts as a hydro- phobic mattress on which a base lies. Second, the main-chain nitrogen of Phe120 forms a hydrogen bond with a nonbridging oxygen atom of the reacting phosphoryl group. (See section VIII.) Thus, even if the side chain of Phe120 did mediate substrate specificity, changing this residue could hamper ca- talysis.

C. One-Dimensional Diffusion

Diffusion is a barrier on the free energy landscape of every bimolecular process.172 The ability to diffuse in one dimension can accelerate the formation of a site-specific interaction within a linear biopolymer by up to 103-fold.173 Such facilitated diffusion is used by transcription factors and restriction endonucleases

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