Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.

摘要: A method is described for the convenient preparation of substantial quantities nucleotide-free p21 or 1:1 complexes with nucleotides other than GDP. The protein has been used kinetic studies binding GDP and GTP, making use fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP -GTP. Stopped-flow have led to formulation a two-step mechanism both involving initial rapid but weak nucleotide followed by relatively slow (10-20 s-1 at 25 degrees C; 3-5 5 C) quasi-irreversible isomerization reaction. By nonequilibrium competition method, guanosine GMP shown interact weakly significantly (dissociation constants 153 29 microM, respectively). presence active site leads marked stabilization against spontaneous denaturation when compared nucleotide- nucleoside-free protein.