Zinc(II)-mediated inhibition of ribonuclease Sa by an N-hydroxyurea nucleotide and its basis.
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 Zn(2+), 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 Zn(2+) to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn(2+). These findings further the development of a new class of ribonuclease inhibitors based on the complex of an N-hydroxyurea nucleotide and zinc(II).
Makarov
Alexander A
AA
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 119991 Moscow, Russia.
Yakovlev
Gennady I
GI
Mitkevich
Vladimir A
VA
Higgin
Joshua J
JJ
Raines
Ronald T
RT
D001665:Binding Sites; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases / Q000737:chemistry; D006918:Hydroxyurea / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry / Q000502:physiology*; D013304:Streptomyces aureofaciens / Q000201:enzymology; D015032:Zinc / Q000737:chemistry*
D007527:Isoenzymes; D009711:Nucleotides; D004722:Endoribonucleases; D012260:Ribonucleases; C077002:ribonuclease Sa3; D015032:Zinc; D006918:Hydroxyurea