A residue to residue hydrogen bond mediates the nucleotide specificity of ribonuclease A.
Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O5 bond of RNA after residues bound in the enzyme's B1 subsite. This subsite binds to cytidine 30-fold more tightly than to uridine and > 10(5)-fold more tightly than to adenine. Structural studies had suggested that the hydroxyl group of Thr45 can interact directly with the base of a bound nucleotide. In contrast, the carboxylate group of Asp83 cannot interact directly with bound substrate but can accept a hydrogen bond from the hydroxyl group of Thr45. To assess the role of the Thr45-Asp83 hydrogen bond in catalysis, T45G, D83A and T45G/D83A RNase A were prepared and their abilities to catalyze the cleavage of various substrates were determined. The results indicate that the side-chain of Asp83 enhances catalysis of reactions in which uridine is bound in the B1 subsite, but that this enhancement relies on the side-chain of Thr45. In contrast, the side-chain of Asp83 does not contribute to catalysis of reactions with cytidine in the B1 subsite. Thermodynamic cycles derived from kinetic parameters for the cleavage of poly(U) indicate that the Thr45-Asp83 interaction contributes 1.2 kcal/mol to transition state stabilization, which is 0.9 kcal/mol greater than its contribution to ground state stabilization. Thus, like many residue-substrate interactions, this residue to residue interaction enhances catalysis by becoming stronger as the reaction approaches the transition state.
delCardayré
S B
SB
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Raines
R T
RT
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D002417:Cattle; D004926:Escherichia coli; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology; D010766:Phosphorylation; D017354:Point Mutation; D011061:Poly A; D011066:Poly C; D011072:Poly U; D011131:Polyribonucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D012265:Ribonucleotides; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011131:Polyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D011061:Poly A; D011072:Poly U; D011066:Poly C; D012259:Ribonuclease, Pancreatic; D000409:Alanine