ACTION OF STAPHYLOCOCCAL NUCLEASE ON SYNTHETIC SUBSTRATES

A number of p-nitrophenyl ester derivatives of deoxythymidine 5′-phosphate can be hydrolyzed by staphylococcal nuclease, with concomitant release of p-nitrophenyl phosphate. The appearance of free p-nitrophenyl phosphate can be continuously monitored spectrophotometrically by following the increase in absorbance at any wavelength in the range 310-350 mμ. The shift of the ultraviolet spectrum of pnitrophenyl phosphate (to higher wavelengths) accompanies conversion of the mono- into the dianionic phosphate species. Consideration of the patterns of cleavage of the synthetic substrates, the structures of various 5′-deoxythymidyl inhibitors, and the relation of these to the observed kinetic constants, permit a partial formulation of the mode of action and specificity of this enzyme. The basic structural unit necessary for recognition as substrate appears to be R-pdT-R′, hydrolysis resulting in the release of R-phosphate. The enzyme appears to be essentially unspecific with respect to R. In the absence of an R substitution, there is strong competitive inhibition of activity. The R′ substituent can be small since the maximal rates of hydrolysis, Kcat, are achieved with a 3′-OH or 3′-acetyl substitution. The nature of the R′ group, however, contributes very significantly to the strength of binding of the substrate (or inhibitor) to enzyme. There is a 200-fold increase in affinity (of substrate and of inhibitor) if R′ is a phosphate rather than a hydroxyl or acetyl group, emphasizing the importance of the phosphate substituents. © 1969, American Chemical Society. All rights reserved.