Fourier transform infrared spectroscopy on the Rap.RapGAP reaction, GTPase activation without an arginine finger.
GTPase activating proteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004) Nature 429, 197-201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP(*) conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras.RasGAP reactions. However, the charge shift toward beta-phosphate observed with RasGAP was also observed for RapGAP. A GDP.P(i) intermediate accumulates in the GAP-catalyzed reaction, because the release of P(i) is eight times slower than the cleavage reaction, and significant GTP synthesis from GDP.P(i) was observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap.RapGAP catalytic machinery compensates for the absence of a cis-Gln by a trans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.
Chakrabarti
Partha P
PP
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Suveyzdis
Yan
Y
Wittinghofer
Alfred
A
Gerwert
Klaus
K
D001120:Arginine / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D020668:rap GTP-Binding Proteins / Q000737:chemistry*
D020690:GTPase-Activating Proteins; D001120:Arginine; D020558:GTP Phosphohydrolases; D020668:rap GTP-Binding Proteins