X hits on this document





9 / 22

Ribonuclease A

Figure 6. Crystalline structure of the active site of RNase A bound to uridine 2,3-cyclic vanadate (U>v). The struc- ture was refined at 2.0 Å from X-ray and neutron diffrac- tion data collected from crystals grown at pH 5.3.164 The side chain of Phe120 and the uracil base are not shown.

reverse of transphosphorylation. Both reactions shown in Figure 5 probably occur via transition states having a pentavalent phosphorus atom. The side chain of Lys41 and the main chain of Phe120 enhance catalysis by stabilizing this transition state (vide infra).

The high-resolution structure of the crystalline complex of RNase A and U>v obtained by joint X-ray/ neutron diffraction analysis has provided invaluable insight into the catalytic mechanism of RNase A. This resolution has been extended to 1.3 Å.167 The active site of this structure is shown in Figure 6. In the active site, the side chains of His12, His119, Lys41, and Gln11, and the mainchain of Phe120 are all proximal to the vanadyl group. The apparent roles of these side chains (and that of Asp121) in catalysis are described below. The main-chain ni- trogen of Phe120 donates a hydrogen bond to a nonbridging oxygen, O3V (N-O3V distance ) 2.9 Å, N-H-O3V angle ) 162°). No data exist on the role of the main-chain nitrogen of Phe120 in catalysis. 164

A. His12 and His119

Histidines were identified as important residues in early work on RNase A. Specifically, haloacetates were shown to carboxymethylate the histidine resi- dues of RNase A.15,228-231 When the proper conditions are effected, only one histidine residue is alkylated in each molecule of RNase A. The rate of the single enzymic carboxymethylation is nearly 104-fold greater than that of free histidine (and greater than that of enzymic carbamoylmethylation), which is consistent with the binding of the anionic haloacetate in the

Chemical Reviews, 1998, Vol. 98, No. 3 1053

cationic active site. The alkylation, which causes a marked decrease in catalytic activity, modifies only His12 or His119.

Catalysis by RNase A has a classic bell-shaped pH-

rate profile.222,232,233

This profile is consistent with a

mechanism that involves two titratable residues, one protonated and the other unprotonated. His12 and His119 are the only residues that need be invoked to explain the pH dependence of catalysis. Recent support for this assignment comes from the semi- synthesis of an RNase A variant containing a 4-fluo- rohistidine residue (7) at both position 12 and posi- tion 119 of RNase A.234 The pH dependence of this

variant is still bell-shaped, but shifted to lower pH. Because 4-fluorohistidine has a lower pKa than does histidine, this perturbation is consistent with both 4-fluorohistidine residues participating in catalysis. These data contradict the conclusion of an earlier study in which substituting 4-flourohistidine at posi- tion 12 of RNase S (see section X) was reported to yield an inactive enzyme that was isostructural with native RNase S. 235

Recombinant DNA techniques have been used to produce RNase A variants in which either His12 or His119 is replaced with an alanine residue.154 The second-order rate constant, kcat/Km, is proportional to the association constant of an enzyme and the rate- limiting transition state during catalysis.236 Elimi- nating the imidazole group of His12 decreases the affinity of the enzyme for this transition state by 104- fold during cleavage of poly(C), UpA, and UpOC6H4- p-NO2. Eliminating the imidazole group of His119 decreased this affinity by 104-fold during cleavage of poly(C) and by almost 104-fold during cleavage of UpA. In contrast, this change had no significant effect on the rate of cleavage of UpOC6H4-p-NO2. Thus, the value of the imidazole group of His119 to catalysis depends on the pKa of the conjugate acid of the leaving groups. The nucleoside leaving groups in poly(C) and UpA have conjugate acids with pKa 14.8 (which is the pKa of CH3OCH2CH2OH237). In contrast, the p-nitrophenolate leaving group of

UpOC 7.14. 6 238

H4-p-NO2 has a conjugate acid with pKa ) Thus, the contribution of His119 to catalysis

decreases when the pKa leaving group decreases.

of the conjugate acid of the This finding is the strongest












nate the addition,

leaving group during RNA cleavage. In Brønsted analyses of catalysis by wild-type





  • -







  • -



are consistent with enzymic reaction.






No analogous evidence for the mechanistic role of His12 is available from kinetic data. One attempt has been made to attain such evidence. If His12 does indeed act as a base, then His12 is likely to contribute less to the enzymic cleavage of 2-deoxy-2-thio-

Document info
Document views101
Page views101
Page last viewedFri Jan 20 02:53:14 UTC 2017