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Ribonuclease A

Figure 7. Putative structure of the transition state during transphosphorylation of UpA by RNase A. The dissociation constant for this complex is KTX e 2 × 10-15 M. 156

Figure 8. Values of kcat/Km for catalysis of the transpho- sphorylation of poly(C) and UpOC6H4-p-NO2 (UpAr) and the hydrolysis of C>p by wild-type RNase A, K41[S- (aminoethyl)cysteine] RNase A, K41R RNase A, and K41A RNase A. Assays were performed at 25 °C in 0.10 M sodium 2-(N-morphilino)ethanesulfonate buffer, pH 6.0, containing NaCl (0.10 M). 157

in the side chain of residue 41 has low activity. These data support a model in which the role of Lys41 is not merely Coulombic, but is to donate a single hydrogen bond to the transition state during cataly- sis.

The role of Lys41 appears to be similar in catalysis of both the transphosphorylation and the hydrolysis reactions (Figure 5). Wild-type RNase A and vari- ants in which Lys41 is replaced by alanine, arginine, and S-(aminoethyl)cysteine were assayed for their abilities to catalyze transphosphorylation [of poly(C) a n d U p O C 6 H 4 - p - N O 2 ] a n d h y d r o l y s i s ( o f U > p ) . T h e r e l a t i v e k c a t / K m v a l u e s a r e s i m i l a r f o r t h e f o u zymes, regardless of the substrate (Figure 8).157 These data are consistent with Lys41 donating a single hydrogen bond in the transition state of both reactions catalyzed by RNase A. A comparison of catalysis by K41A RNase A and the wild-type enzyme shows that this hydrogen bond lowers the free energy of the rate-limiting transition state for poly(C) cleav- age by 5 kcal/mol. In contrast to catalysis by the wild-type enzyme, a change in covalency limits catalysis by K41A RNase A. r e n - 156

Chemical Reviews, 1998, Vol. 98, No. 3 1055

C. Asp121

In native RNase A, Asp121 can interact with His119, the acid in the catalysis of RNA cleavage. The interaction between His119 and Asp121 defines a motif known as the catalytic dyad, in which a histidine residue that mediates general acid/base catalysis forms a hydrogen bond with an aspartate residue. This motif bears a striking resemblance to the conserved motif known as the catalytic triad found in serine proteases. (For reviews, see refs 260 and 261.)

Several attempts have been made to determine the role of aspartate in the catalytic dyad of RNase A. In one study, Asp121 was replaced with asparagine in a semisynthetic enzyme. This semisynthetic ri- bonuclease, RNase(1-118)(111-124),262,263 consists of a noncovalent complex between residues 1-118 of RNase A (obtained from proteolytic digestion of RNase A), and an overlapping synthetic peptide composed of the 14 C-terminal residues of RNase A, except with Asp121 replaced by an asparagine resi- due. The D121N semisynthetic variant has ap- proximately 5% of the catalytic activity of the analo- gous wild-type semisynthetic enzyme.264 These data are difficult to interpret, however, because the three- dimensional structure D121N semisynthetic variant deviates from that of RNase(1-118)(111-124).265-267

Site-directed mutagenesis has been used to replace Asp121 with glutamate, asparagine, and alanine residues.36,153,268-270 The glutamate variant has ap- proximately 17% of the activity of the wild-type enzyme for C>p hydrolysis.153 The crystalline struc- tures of the other two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R factor of 0.18.270 The alterations do not perturb the conformation of the enzyme. In the structure of D121N RNase A, Nδ rather than Oδ of Asn121 faces His119. The values of kcat/Km and kcat for transpho- sphorylation of UpA and poly(C) are reduced by 101- fold (D121N) and 102-fold (D121A).270 The values of kcat/Km and kcat for hydrolysis of U>p are reduced by 3-fold (D121N) and 10-fold (D121A). The alterations do not otherwise effect the pH-rate profiles for hydrolysis. These decreases are far less than than those observed for analogous variants of serine


Overall, the His‚‚‚Asp hydrogen

bond in the active site of RNase A has a significant but not substantial role in catalysis. This role is likely to position the proper tautomer of His119.

A major difference betweeen Asp121 of RNase A and the aspartate residue in the catalytic triad of serine proteases is solvent exposuresAsp 121 is more accessible to solvent. In native RNase A, Asp121 can form hydrogen bonds with solvent water. It is therefore not surprising that the hydrogen bond in the His‚‚‚Asp catalytic dyad of RNase A plays a less significant role than do the analogous hydrogen bonds in serine proteases. 276,277

Replacing Asp121 with an asparagine or alanine

residue results in a loss of conformational stability a t p H 6 . 0 o f G m ) - 2 . 0 k c a l / m o l , f r o m a t o t a l G m ) 9 . 0 k c a l / m o l . 2 6 9 T h i s l o s s i s s i m i l a r i n m a g n i to the loss of transition-state binding during catalysis t u d e










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