Further studies on the specificity of the minor ribonuclease from Aspergillus saitoi.
In order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25 degrees. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The Ki values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribonucleosides was in the order dT, dA, dG, and dC.
D001230:Aspergillus / Q000201:enzymology*; D003853:Deoxyribonucleosides / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004720:Endonucleases / Q000378:metabolism*; D007700:Kinetics; D012260:Ribonucleases / Q000378:metabolism*; D012263:Ribonucleosides / Q000494:pharmacology; D012265:Ribonucleotides / Q000378:metabolism; D013329:Structure-Activity Relationship
D003853:Deoxyribonucleosides; D003854:Deoxyribonucleotides; D012263:Ribonucleosides; D012265:Ribonucleotides; D004720:Endonucleases; D012260:Ribonucleases