The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the P0, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the P0 and P2 subsites of RNase A is characterized in detail. Lys66 (P0 subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of kcat and Km for poly(C) cleavage were affected by altering the P0 and P2 subsites. The kcat/Km values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the P0 and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the P0 and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the P0 subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the P0 or P2 subsite. The logarithm of Kd is a linear function of the logarithm of [Na+] over the range 0.018 M

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The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid. Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously [Fisher, B. M., Ha, J.-H., and Raines, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail. A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively. There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site. These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic pKa values. Finally, the affinity of 3'-UMP for wild-type RNase A and the K7A/R10A/K66A variant at 0.018 and 0.142 M Na+ was determined by isothermal titration calorimetry. 3'-UMP binds to the variant protein with 5-fold weaker affinity at 0.018 M Na+ and 3-fold weaker affinity at 0.142 M Na+ than it binds to wild-type RNase A. Together these data demonstrate that long-range Coulombic interactions are an important feature in catalysis by RNase A.

The interaction between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extends beyond the scissile bond. Enzymic subsites interact with the bases and the phosphoryl groups of a bound substrate. We evaluated the four cationic residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 were replaced individually by an alanine residue, and the resulting enzymes were assayed as catalysts of poly-(cytidylic acid) (poly(C)) cleavage. The values of K m and k cat /K m for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the transition state was uniformOK m increased by 15-fold and k cat /K m decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of K d for complexes with oligonucleotides of different length. This contribution was dependent on salt concentration, as expected from a coulombic interaction between a cationic side chain and an anionic phosphoryl group. Together, these data indicate that Arg-85 interacts with a particular phosphoryl group of a bound nucleic acid. We propose that Arg-85 comprises a new distal subsite in RNase AOthe P(؊1) subsite.The efficiency of enzymatic catalysts is a source of continued interest and inspiration as molecular scientists strive to design new catalysts. A distinguishing characteristic of enzymic catalysts is that they bind to their substrates (1, 2). Binding energy is necessary to compensate for the loss of translational and rotational entropy and for any destabilization of the substrate required to reach the transition state (3, 4). Multivalent contacts between an enzyme and substrate provide much of this required binding energy. Indeed, many enzymes that cleave polymeric substrates have subsites that interact with monomeric units of the substrate.Bovine pancreatic ribonuclease A (RNase A; 1 EC 3.1.27.5) is a classic model for revealing the physical, chemical, and biological properties of enzymes (5, 6). RNase A is a 13.7-kDa endoribonuclease that binds RNA in a cationic cleft and cleaves on the 3Ј-side of pyrimidine residues. The cleft contains subsites (B1, B2, and B3) that interact specifically with bases and subsites (P0, P1, and P2) that interact with phosphoryl groups (7,8). The specificity of RNase A for pyrimidine bases is because of exclusion of the larger purine bases from the B1 subsite (9). The B2 and B3 subsites prefer to bind purine bases. His-12, His-119, and Lys-41 of the P1 subsite are the residues most central to the catalytic function of the enzyme. The amino acid residues that comprise the P0 (Lys-66) and P2 (Lys-7 and Arg-10) subsites increase the affinity with which the substrate binds to the enzyme and participate indirectly in catalysis (10 -12).Some data portend the existence of additional RNase A binding sites beyond those characterized previously. Three-dimensional structures derived from x-ray diffraction analyses reveal a line of cationic res...

Background Pregnant women have an elevated risk of illness and hospitalisation from influenza. Pregnant women are recommended to be prioritised for influenza vaccination during any stage of pregnancy. The risk of seasonal influenza varies substantially throughout the year in temperate climates; however, there is limited knowledge of how vaccination timing during pregnancy impacts the benefits received by the mother and foetus. Objectives To compare antenatal vaccination timing with regard to influenza vaccine immunogenicity during pregnancy and transplacental transfer to their newborns. Methods Studies were eligible for inclusion if immunogenicity to influenza vaccine was evaluated in women stratified by trimester of pregnancy. Haemagglutination inhibition (HI) titres, stratified by trimester of vaccination, had to be measured at either pre‐vaccination and within one month post‐vaccination, post‐vaccination and at delivery in the mother, or in cord/newborn blood. Authors searched PubMed, Scopus, Web of Science and EMBASE databases from inception until June 2016 and authors of identified studies were contacted for additional data. Extracted data were tabulated and summarised via random‐effect meta‐analyses and qualitative methods. Results Sixteen studies met the inclusion criteria. Meta‐analyses found that compared with women vaccinated in an earlier trimester, those vaccinated in a later trimester had a greater fold increase in HI titres (1.33‐ to 1.96‐fold) and higher HI titres in cord/newborn blood (1.21‐ to 1.64‐fold). Conclusions This review provides comparative analysis of the effect of vaccination timing on maternal immunogenicity and protection of the infant that is informative and relevant to current vaccine scheduling for pregnant women.