Structures and Functions of Ribonucleases

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.