Ribonuclease Sa (RNase Sa) is a secretory ribonuclease from Streptomyces aureofaciens. Herein, 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is shown to be a competitive inhibitor of catalysis by RNase Sa. Inhibition is enhanced by nearly 10-fold in the presence of Zn2+, which could coordinate to the N-hydroxyurea group along with enzymic residues. The carboxylate of Glu54 is the putative base that abstracts a proton from the 2' hydroxyl group during catalysis of RNA cleavage by RNase Sa. Replacing Glu54 with a glutamine residue has no effect on the affinity of N-hydroxyurea 1 for the enzyme, but eliminates the zinc(II)-dependence of that affinity. These data indicate that an N-hydroxyurea nucleotide can recruit Zn2+ to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn2+. These findings further the development of a new class of ribonuclease inhibitors based on the complex of an N-hydroxyurea nucleotide and zinc(II). (C) 2004 Elsevier Inc. All rights reserved.