1056 Chemical Reviews, 1998, Vol. 98, No. 3
His‚‚‚Asp catalytic dyad is to enhance the conforma- tional stability of the enzyme. The pH dependencies of the conformational stabilities of the wild-type, D121N, D121A, and H119A enzymes reveal that the pKa of Asp121 is 2.7 in native wild-type RNase A but 3.6 in the denatured enzyme. The side chain of His119 is largely responsible for this change in pKa.
The kinetics of catalysis by D121N RNase A and D121A RNase A illuminate another aspect of the mechanism of RNase A. The side chain of His119 can occupy two conformations that differ by rotation about the CR-C bond. In one of these conformations (position A), the side chain of His119 forms a hydro- gen bond with the side chain of Asp121. In the other conformation (position B), the side chain of His119 forms a hydrogen bond with solvent. In the three- dimensional structures of RNase A bound to d(CpA) and cytidylyl(2′f5′)adenosine, the adenine base pre- vents His119 from being in position B.79,278 Thus, His119 must act from position A during catalysis of transphosphorylation. But structural data show that His119 could act from either position A or position B during catalysis of hydrolysis. Indeed, it has been suggested that the AhB equilibrium evolved to enable transphosphorylation to occur with His119 in
A and B.279
hydrolysis to occur with Yet, RNase A with an
His119 in aspartate,
or alanine abilities to
residue in position 121 catalyze hydrolysis. 269
His119 during catalysis of hydrolysisshydrolysis occur with His119 in position A.
X-ray diffraction analyses show that the side chain
of Gln11 can form a hydrogen bond to a substrate, substrate analogue, phosphate ion, or sulfate ion bound in the active site of RNase A. (For a review of these analyses, see ref 82.) 1H NMR spectroscopy provides further evidence for this interaction, as large changes in the NH1 and NH2 resonances of Gln11 are observed upon binding of pyrimidine nucle- otides.280 In the high-resolution structure of RNase A complexed with U>v (Figure 6), the side-chain nitrogen of Gln11 forms a hydrogen bond with the n o n b r i d g i n g o x y g e n O 1 V ( N δ 2 - O 1 V d i s t a n c e ) 2 . 6 Å , N δ 2 - H - O 1 V a n g l e ) 1 4 0 ° ) . 1 6 4 A s t u d y o f s e m thetic variants of RNase S (see section X) having various residues at position 11 have also ascribed a significant role for Gln11 in catalysis.281 Together, these data portend an important role for Gln11 in i s y n -
the catalytic mechanism of RNase A.
The role of Gln11 in catalysis by RNase A has been probed by creating variants in which this residue is replaced with alanine, glutamine, and histidine. The results show that Gln11 does not stabilize the rate-limiting transition state during catalysis by RNase A. Rather, Gln11 serves to increase the free energy of the enzyme‚substrate complex. 37
The destabilization of the enzyme‚substrate com- plex may be an obligatory event in the evolution of
and can arise from a
variety of molecular scenarios. In RNase A, the increase in the free energy of the Michaelis complex appears to be due (at least in part) to a binding
interaction that reduces nonproductive binding. In the absence of the side chain of Gln11, the active site is more likely to bind an RNA molecule with its phosphoryl group in an improper conformation for in- line attack by the 2′-hydroxyl group. The increase in the number of substrate binding modes causes a decrease in the value of kcat and an identical decrease in the value of Km, such that the value of kcat/Km is unchanged.236 This effect is most dramatic in the turnover of UpOC6H4-p-NO2 by Q11A RNase A. This substrate, unlike poly(C) or UpA, cannot interact with enzymic subsites on both sides of the scissile bond, making its proper alignment problematic. The values of both kcat and Km for the cleavage of UpOC6H4-p-NO2 by Q11A RNase A are 102-fold lower than those for the cleavage of UpOC6H4-p-NO2 by the wild-type enzyme. Thus, a hydrogen bond between the side chain of Gln11 and a phosphoryl oxygen appears to enhance catalysis in a subtle mannersby orienting the substrate so as to prevent it from binding in a nonproductive mode.
IX. Reaction Energetics
The energetics of catalysis by RNase A are not yet characterized completely. Like proteases, ribonu- cleases catalyze exergonic reactions. Monitoring the reverse of the transphosphorylation and hydrolysis reactions is difficult. The revelation of the reaction energetics of ribonuclease catalysis is therefore more challenging than is that of enzymes such as triose- phosphate isomerase and proline isomerase,284 which catalyze the relatively isogonic interconversion of a single substrate and a single product. Regardless, progress has been made with RNase A.
A. Transphosphorylation versus Hydrolysis
RNase A catalyzes both the transposphorylation of RNA to form a 2′,3′-cyclic phosphodiester intermedi- ate and hydrolysis of this cyclic intermediate to form a 3′-phosphomonoester (Figure 5).285,286 31P NMR spectroscopy287,288 has been used to monitor in a continuous assay the extent to which the 2′,3′-cyclic phosphodiester intermediate accumulates during ca- talysis by RNase A and small molecules.183 31P NMR spectra show that the cyclic intermediate accumu- lates during catalysis by RNase A. The enzyme releases rather than hydrolyzes most of the 2′,3′- cyclic phosphodiester product of RNA transphospho- rylation, a result in accord with earlier chromato-
In contrast, the cyclic
intermediate does not accumulate during catalysis by hydroxide ion or imidazole buffer.183 In the presence of these small-molecule catalysts, hydrolysis of the cyclic intermediate is faster than transphos- phorylation of RNA.
A trapping experiment has been used to evaluate the “throughput” of the reaction catalyzed by RNase A. [5,6-3H]UpA was incubated with RNase A in the presence of excess unlabeled uridine 2′,3′-cyclic phos- phodiester, which dilutes the specific radioactivity of any released cyclic intermediate. Only 0.1% of the RNA substrate was found to be both transphospho- rylated and hydrolyzed without dissociating from the