enzyme. These results suggest that RNase A has evolved primarily to catalyze transphosphorylation rather than hydrolysis. [To denote this preference, perhaps RNase A should be referred to (once again15) as an “RNA depolymerase”.] Many textbooks (cf., refs 250 and 289-292) incorrectly picture the mechanism of RNA hydrolysis by RNase A as proceeding in one two-step process rather than in two one-step pro- cesses (Figure 5). 182,183
The result of the throughput experiment has an important implication for the mechanism of the reaction catalyzed by RNase A. The imidazole group of His12 acts as a base in the transphosphorylation reaction and an acid in the hydrolysis reaction. The imidazole group of His119 has a complementary role, acting as an acid in the transphosphorylation reac- tion and a base in the hydrolysis reaction. After catalysis of transphosphorylation, each histidine residue in the active site of RNase A is protonated appropriately to catalyze hydrolysis of the bound cyclic intermediate. After hydrolysis of this sub- strate, each histidine residue is returned to its initial protonation state, completing the catalytic cycle. But RNase A short-circuits this cycle by releasing rather than hydrolyzing the cyclic intermediate. Thus, RNase A has an iso mechanism293,294 in which the protonation states of the unliganded enzyme are interconverted by a pathway that does not involve substrate molecules.
B. Rate Enhancement
The products of the uncatalyzed cleavage of UpA are the same as those in the enzyme-catalyzed reaction.156 The identity of these reaction products is consistent with the uncatalyzed and catalyzed transphosphorylation reactions proceeding by the same mechanism. If a reaction does proceed by the same mechanism in the absence and presence of an enzyme, then the ratio of kcat/Km for the enzyme- catalyzed reaction to kuncat for the uncatalyzed reac- tion provides a measure of the affinity of the enzyme for the rate-limiting transition state during cataly- sis.295 At pH 6.0 and 25 °C, RNase A catalyzes the transphosphorylation of UpA with a kcat/Km of 2.3 × 106 M-1 s-1.146 Under identical conditions, the un- catalyzed rate of UpA transphosphorylation, mea- s u r e d b y f o l l o w i n g t h e c l e a v a g e o f [ 5 , 6 - 3 H ] U p [ 3 , 5 , 8 - -9 3 s-1 H]A for several weeks, corresponds to t1/2 ) 4 y). (which is 5 The 10 × 156 dissociation constant
transphosphorylation (kcat/Km) ) 2 × 10-15 transition state may
of UpA is therefore KTX ) kuncat/ M. Because the rate-limiting not involve a change in cova-
dissociation constant of the chemical transition state for
enzyme bound to the P-O5′ bond cleavage.
What is the origin of the affinity of RNase A for the chemical transition state? Replacing Lys41 with an alanine residue removes a potential hydrogen- bond donor from the active site of RNase A. It is the ability of this residue to donate a hydrogen bond that enhances catalysis.155 The loss of a hydrogen bond from residue 41 costs the enzyme 105-fold in rate acceleration. Similarly, replacing His12 or His119,
Chemical Reviews, 1998, Vol. 98, No. 3 1057
(- - -)
RNase A-catalyzed (s) transphosphorylation of UpA (left) and hydrolysis of U>p (right). Free energies of activation were calculated for the reaction at pH 6.0 and 25 °C with t h e e q u a t i o n : ∆ G q ) - R T l n [ k h / ( k b T ) ] a n d t h e v a l u e s o f k c a t / K m 1 4 6 a n d k c a t 1 5 6 f o r U p A t r a n s p h o s p h o r y l a t i o n , k c a t / K m f o r U > p h y d r o l y s i s , 3 7 a n d k u n c a t f o r C > p h y d r o l y free energy of uridine 3′-phosphate (3′-UMP) relative to that of U>p was calculated for ther reaction at pH 6.0 and 25 °C with the equation: ∆G° ) -RT ln K, where K ) 1.0 × 103.233 The free energies for the RNase A-catalyzed reactions are drawn for a standard state of 0.1 mM, which is the concentration of RNase A in the bovine pancreas.2 The uncatalyzed hydrolysis of U>p also produces uridine s i s . 2 9 7 T h e
2′-phosphate in a reaction that is not shown.
the base and acid in catalysis (Figure 5), slows catalysis by 104- to 105-fold.154 Finally, the B2 subsite of RNase A is also significant contributor to catalysis. This subsite, which interacts with the base of the residue that is part of the scissile phosphodiester bond, is composed of Asn71 and Glu111 (Figure 2). The values of kcat/Km for the RNase A catalyzed transphosphorylation of substrates with different leaving groups decrease in the order: adenosine > guanosine > cytidine > uridine > methanol.16 CpA 141
transphosphorylated by RNase A with k 106 M-1 s-1; CpOMe with kcat/Km ) 250 M cat -1 /K s m -1 296 ) 3
If CpA interacts and CpOMe does
most strongly not interact at
with the B2 pocket all, then the binding
of adenosine acceleration.
to the B2 subsite provides a 104-fold rate Thus, four factors (Lys41, His12, His119,
104-fold in rate enhancement. Because the rate enhancement is 3 × 1011, these factors contribute independently to catalysis.
The free energies for the two steps in the hydrolysis of RNA can be derived from available data (Figure 9233,297).156 At pH 6.0 and 25 °C, the intrinsic kinetic barrier for cleaving a P-O5′ bond in RNA is almost identical to that for hydrolyzing the P-O2′ or P-O3′ bond in a nucleotide 2′,3′-cyclic phosphodiester. Ap- parently, the proximity of the 2′-hydroxyl group to the phosphorus atom in RNA and the strain298-301 (or poor solvation302) inherent in a nucleotide 2′,3′-cyclic phosphodiester contribute equally to an enhanced rate of decomposition. These phosphodiester bonds are far less stable than are those in DNA, which suffer cleavage at a 3 × 104-fold lower rate. Together, kinetic data on the cleavage of the P-O bond in RNA156 and DNA303 reveal that each proximal 2′-hydroxyl group of RNA has an effective concentra- tion of 2 × 106 M () 3 × 104 × 55 M). 303 5′