Insight into catalysis of a unique GTPase reaction by a combined biochemical and FTIR approach.
Rap1 and Rap2 are the only small guanine nucleotide-binding proteins of the Ras superfamily that do not use glutamine for GTP hydrolysis. Moreover, Rap1GAP, which stimulates the GTPase reaction of Rap1 10(5)-fold, does not have the classical "arginine finger" like RasGAP but presumably, introduces an asparagine residue into the active site. Here, we address the requirements of this unique reaction in detail by combining various biochemical methods, such as fluorescence spectroscopy, stopped-flow and time-resolved Fourier transform infrared spectroscopy (FTIR). The fluorescence spectroscopic assay monitors primarily protein-protein interaction steps, while FTIR resolves simultaneously the elementary steps of functional groups labor-free, but it is less sensitive and needs higher concentrations. Combining both methods allows us to distinguish weather mechanistic defects caused by mutation are due to affinity or due to functionality. We show that several mutations of Asn290 block catalysis. Some of the mutants, however, still form a complex with Rap1*GDP in the presence of BeF(x) but not AlF(x), supporting the notion that fluoride complexes are indicators of the ground versus transition state. Mutational analysis also shows that Thr61 is not required for catalysis. While replacement of Thr61 of Rap1 by Leu eliminates GTPase activation by Rap1GAP, the T61A and T61Q mutants have only a minor effect on catalysis, but change the relative rates of cleavage and (P(i)(-)) release. While Rap1GAP(N290A) is completely inactive on wild-type Rap1, it can act on Rap1(T61Q), arguing that Asn290 in trans has a role in catalysis similar to that of the intrinsic Gln in Ras and Rho. Finally, since FTIR works at high, and thus mostly saturating, concentrations, it can clearly separate effects on affinity from purely catalytic modifications, showing that Arg388, conserved between RapGAPs and mutated in the homologous RheBGAP Tuberin, affects binding affinity severely but has no effect on the cleavage reaction itself.
Chakrabarti
Partha P
PP
Abteilung Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie, D-44227 Dortmund, Germany.
Daumke
Oliver
O
Suveyzdis
Yan
Y
Kötting
Carsten
C
Gerwert
Klaus
K
Wittinghofer
Alfred
A
D001120:Arginine / Q000235:genetics / Q000502:physiology; D001216:Asparagine / Q000235:genetics / Q000502:physiology; D001669:Biochemical Phenomena; D001671:Biochemistry; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli; D005459:Fluorides / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared; D013912:Threonine / Q000235:genetics / Q000502:physiology; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D050505:Mutant Proteins; C484658:RAP1GAP protein, human; D013912:Threonine; D001216:Asparagine; D001120:Arginine; D020827:rap1 GTP-Binding Proteins; D005459:Fluorides