Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O 5′ bonds in RNA. Structural analyses had suggested that the active-site lysine residue (K41) may interact preferentially with the transition state for covalent bond cleavage, thus facilitating catalysis. Here, site-directed mutagenesis and semisynthesis were combined to probe the role of K41 in the catalysis of RNA cleavage. Recombinant DNA techniques were used to replace K41 with an arginine residue (K41R) and with a cysteine residue (K41C), which had the only sulfhydryl group in the native protein. The value of k cat /K m for cleavage of poly(C) by K41C RNase was 10 5 -fold lower than that by the wild-type enzyme. The sulfhydryl group of K41C RNase A was alkylated with 5 different haloalkylamines. The value of k cat /K m for the resulting semisynthetic enzymes and K41R RNase A were correlated inversely with the values of pK a for the side chain of residue 41. Further, no significant catalytic advantage was gained by side chains that could donate a second hydrogen bond. These results indicate that residue 41 donates a single hydrogen bond to the ratelimiting transition state during catalysis.

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O 5′ bond in RNA. Although this enzyme has been the object of much landmark work in bioorganic chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k cat /K m for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k cat /K m for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC 6 H 4 -p-NO 2 by wild-type RNase A were found to be independent of glycerol concentration. Yet, UpA cleavage by the wild-type and mutant enzymes was found to have the same dependence on sucrose concentration, indicating that catalysis of UpA cleavage by RNase A is limited by desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k cat = 1.4 × 10 3 s −1 and k cat /K m = 2.3 × 10 6 M −1 s −1 at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5, H]Up [3,5, H]A cleavage was found to be k uncat = 5 × 10 −9 s −1 (t 1/2 = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10 11 -fold by binding to the transition state for P-O 5′ bond cleavage with a dissociation constant of <2 × 10 −15 M.

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Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2′,3′-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 10 5 -fold in their k cat /K m values for catalysis, but <40-fold in their K i values for inhibition of catalysis by U>v. Plots of logK i vs log(K m /k cat ) for three distinct substrates [poly(cytidylic acid), uridine 3′-(p-nitrophenyl phosphate), and cytidine 2′,3′-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K i for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.

The facile modulation of biological processes is an important goal of biological chemists. Here, a general strategy is presented for controlling the catalytic activity of an enzyme. This strategy is demonstrated with ribonuclease A (RNase A), which catalyzes the cleavage of RNA. The side-chain amino group of Lys41 donates a hydrogen bond to a nonbridging oxygen in the transition state for RNA cleavage. Replacing Lys41 with a cysteine residue is known to decrease the value of k(cat)/K(m) by 10(5)-fold. Forming a mixed disulfide between the side chain of Cys41 of K41C RNase A and cysteamine replaces the amino group and increases k(cat)/K(m) by 10(3)-fold. This enzyme, which contains a mixed disulfide, is readily deactivated by dithiothreitol. Forming a mixed disulfide between the side chain of Cys41 and mercaptopropyl phosphate, which is designed to place a phosphoryl group in the active site, decreases activity by an additional 25-fold. This enzyme, which also contains a mixed disulfide, is reactivated in the presence of dithiothreitol and inorganic phosphate (which displaces the pendant phosphoryl group from the active site). An analogous control mechanism could be installed into the active site of virtually any enzyme by replacing an essential residue with a cysteine and elaborating the side chain of that cysteine into appropriate mixed disulfides.