Mode of hydrolysis of diribonucleoside monophosphates by phosphodiesterase-phosphomonoesterase of Fusarium moniliforme.
The products derived from the degradation of the sixteen possible diribonucleoside monophosphates (NpN') by Fusarium phosphodiesterase-phosphomonoesterase were analyzed by means of thin layer chromatography. The analysis showed that NpN' was first cleaved into nucleoside N and 5'-nucleotide pN', which was then dephosphorylated to yield nucleoside N'. The dephosphorylation was fast when N' was adenosine or cytidine but slow when N' was guanosine or uridine. The cleavage reaction was followed by measuring the increase of absorbance due to hyperchromicity, and the kinetic constants, Km and kcat, were determined for the sixteen dinucleoside phosphates. The Km value was higher, for a given N, when N' was a pyrimidine nucleoside than when N' was a purine nucleoside. For a given N', uridine as N gave the highest Km value and adenosine gave the lowest one. The kcat value was the highest, for a given N, when N' was cytidine. For a given N', uridine as N gave by far the lowest kcat value. These results can be interpreted in terms of two binding sites on the enzyme with different base preferences. Comparison of kcat/Km values suggested that the base of nucleoside N plays an important role in determining whether a dinucleoside phosphate is a good substrate of the enzyme. The dinucleoside phosphates with uridine as N were found to be particularly poor substrates of the enzyme.
D002855:Chromatography, Thin Layer; D015226:Dinucleoside Phosphates; D005656:Fungal Proteins; D005670:Fusarium / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009705:Nucleosides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*
D015226:Dinucleoside Phosphates; D005656:Fungal Proteins; D009705:Nucleosides; D009841:Oligonucleotides; D010744:Phosphoric Monoester Hydrolases; C019047:phosphodiesterase-phosphomonoesterase; D010727:Phosphoric Diester Hydrolases