Structural determinants of the uridine-preferring specificity of RNase PL3.
RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.
Vicentini
A M
AM
Friedrich Miescher-Institut, Basel, Switzerland.
Kote-Jarai
Z
Z
Hofsteenge
J
J
D000818:Animals; D001665:Binding Sites; D003562:Cytidine / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011066:Poly C / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013912:Threonine / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C013907:cytidylyl adenosine; D011072:Poly U; D013912:Threonine; D011066:Poly C; C009628:uridylyl-(3'-5')-adenosine; D003562:Cytidine; D004722:Endoribonucleases; C062547:ribonuclease PL(3); D014529:Uridine