SUBSTRATE AND PRODUCT STRUCTURAL REQUIREMENTS FOR BINDING OF NUCLEOTIDES TO H-RAS P21 - THE MECHANISM OF DISCRIMINATION BETWEEN GUANOSINE AND ADENOSINE NUCLEOTIDES

The interaction of the protein product of the H-ras oncogene with a se ries of nucleoside di- and triphosphates has been examined to investig ate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in a simple filter binding assay to give semiqua ntitave estimates of their affinities. For more weakly binding nucleot ides or to obtain quantitative data, a transient kinetic method was us ed which was based on determination of the association and dissociatio n rate constants. The results obtained indicate that substantial modif ication of the sugar or phosphate structure is tolerated with little o r moderate loss of affinity, but that large losses in affinity occur o n modification of the base structure. In particular, replacing the gua nine by an adenine residue leads to a dramatic loss of affinity. Thus, discrimination against ATP and ADP is very high (relative affinities of ATP and GTP 1:10(7)). This is due not only to loss of positive (sta bilizing) interactions, but especially to the introduction of negative ones.