STRUCTURAL DIFFERENCES IN THE MINIMAL CATALYTIC DOMAINS OF THE GTPASE-ACTIVATING PROTEINS P120(GAP) AND NEUROFIBROMIN

The kinetic properties for the enzymatic stimulation of the GTPase rea ction of p21(ras) by the two GTPase-activating proteins (GAPs) p120(GA P) and neurofibromin are different. In order to understand these diffe rences and since crystallization attempts have only been successful wi th truncated fragments, structure/function requirements of the catalyt ic core of these proteins were investigated. Differences in size of th e minimal catalytic domains of these two proteins mere found as determ ined by limited proteolysis. The minimal catalytic domain has a molecu lar mass of 30 kDa in the case of p120(GAP) and of 26 kDa in the case of neurofibromin. Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs. The C te rmini of these fragments are identical, whereas the N-terminal part of the minimal p120(GAP) domain is 47 amino acids longer. These newly id entified minimal catalytic fragments were as active in stimulating GTP ase activity toward p21(ras) as the corresponding larger fragments GAP -334 and NF1-333 from which they had been generated via proteolytic di gestion. Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catal ytic activity. In our hands this protein is unstable and has no cataly tic activity. Thus, we believe that we have defined the true minimal d omains of p120(GAP) (GAP-273, residues Met(714)-His(986)) and neurofib romin (NF1-230, residues Asp(1248)-Phe(1477)), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity .