Ribonuclease 4, an evolutionarily highly conserved member of the superfamily.

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a Kd of 4 x 10(-15) M.

A single amino acid substitution changes ribonuclease 4 from a uridine-specific to a cytidine-specific enzyme.

The structural features underlying the strong uridine specificity of ribonuclease 4 (RNase 4) are largely unknown. It has been hypothesized that the negatively charged alpha-carboxylate is close to the pyrimidine binding pocket, due to a unique C-terminal deletion. This would suppress the cleavage of cytidine-containing substrates [Zhou, H.-M., and Strydom, D. J. (1993) Eur. J. Biochem. 217, 401-410]. Replacement of the alpha-carboxylate by an alpha-carboxamide in a fragment complementation system decreased both (kcat/Km)CpA and (kcat/Km)UpA , thus refuting the hypothesis. However, model building showed that the deletion allowed the side chain of Arg-101 to reach the pyrimidine binding pocket. From the 386-fold reduction in (kcat/Km)UpA in RNase 4;R101N, it is concluded that this residue functions in uridine binding, analogous to Ser-123 in RNase A. In addition, it may have an effect on Asp-80. The 2-fold increase in (kcat/Km)CpA in the mutant R101N and the close proximity of the side chains of Arg-101 and Asp-80 suggested that the latter could be involved in suppressing CpA catalysis. The substrate specificity of RNase 4;D80A was completely reversed: (kcat/Km)UpA decreased 159-fold, whereas (kcat/Km)CpA increased 233-fold. The effect on CpA was unexpected, because the corresponding residue in bovine pancreatic RNase A (Asp-83) hardly affects cytidine-containing substrates. Furthermore, the residue is conserved in nearly all sequences of mammalian RNase 1. Thus, an evolutionary highly conserved residue does not necessarily function in the same way in homologous enzymes. A model, which proposes that the structure of RNase 4 has been optimized to permanently fix the position of Asp-80 and impede its movement away from the pyrimidine binding pocket, is presented.

Protease activity of 1,10-phenanthroline-copper(I). Targeted scission of the catalytic site of carbonic anhydrase.

To investigate the mechanism of scission of proteins by the chemical cleaving agent 1,10-phenanthroline-copper, the active sites of human carbonic anydrase I and bovine carbonic anhydrase II have been targeted for cleavage by a tight binding sulfonamide inhibitor tethered to the metal complex. The inhibitor-phenanthroline-copper conjugate binds to the carbonic anhydrases with sub-micromolar Kd's and, upon addition of a reducing agent, causes scission specifically within the active site of the enzymes to yield a discrete set of cleavage fragments. N- and C-terminal sequencing and mass spectrometric analysis of several fragments indicate that the C-terminal cleavage fragments have free amino groups at their N termini, thereby allowing facile location of the cut sites through standard Edman degradation. The N-terminal cleavage fragments do not have a free carboxyl group at their C termini. It is proposed that scission occurs by abstraction of H at Calpha, followed by oxidation at Calpha by the neighboring cupric ion and cleavage of the Calpha-C(O) bond to give an N-terminal fragment containing a C-terminal acyl amide, and an unstable C-terminal fragment containing an N-terminal isocyanate group which undergoes hydrolysis to a free amino terminus. Modeling of the inhibitor-phenanthroline-copper conjugate within the active site of human carbonic anhydrase I shows that the sites of cleavage that have been identified are fully consistent with the available structural data.

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.