Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTPhydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10 5 -fold by Rap1GAP and has a k cat of 6 s ؊1 at 25°C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V max of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25-and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10 6 -fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.
McrBC is a GTP-dependent restriction endonuclease of E. coli K12, selectively directed against DNA containing modified cytosine residues. McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB. Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites. (ii) Strong DNA binding (K(ass) approximately 10(7)M[-1]) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-). (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP. (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates. (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding. (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.
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