The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a >1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (-100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.The cellular ras genes encode plasma membrane-associated proteins (p2ls) of 189 amino acids which bind GDP and GTP with high affinity and display a low level of GTPase activity (see reference 1 for a review). These properties of p21 are analogous to the G proteins, which transduce signals from a variety of cell surface receptors to enzymes which affect metabolism of second messengers (see reference 11 for a review). It is therefore an attractive hypothesis that p2is function analogously to the G proteins to affect second messenger pathway(s) which can result in abnormal cell proliferation.One approach to understanding the function of p21 has been comparative analysis of the biological and biochemical activities of the products of mutated ras genes. Activation of the transforming potential of ras genes in human tumors has been found to result from single amino acid substitutions at positions 12, 13, and 61 (1). The viral rasH and rasK genes also contain activating mutations at position 59 (1). Activating mutations at positions 12 and 61 decrease the GTPase activity of p21 approximately 10-fold (6, 10, 14, 15, 18). Since GTP hydrolysis mediates physiological deactivation of the G proteins, these results are consistent with the Gprotein analogy. However, reduced GTPase does not correlate quantitatively with the transforming ...