gamma-phosphate protonation and pH-dependent unfolding of the Ras.GTP.Mg2+ complex: a vibrational spectroscopy study.
The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the gamma-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes. Isotope-edited IR studies of the Ras.GTP.Mg(2+) complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma-phosphate is not protonated at pH > or = 3.3, indicating that the active site decreases the gamma-phosphate pK(a) by at least 1.1 pK(a) units compared with solution. Amide I studies show that the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes (3.7 and 4.8, respectively) reveal that the enzyme-gamma-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras.GTP.Mg(2+) complex provides an alternative interpretation of the data that had been used to support the gamma-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.
Cheng
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H
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461-1926, USA.
Sukal
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S
Callender
R
R
Leyh
T S
TS
D004926:Escherichia coli / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009154:Mutation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011522:Protons; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics; D013997:Time Factors; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
D010710:Phosphates; D011522:Protons; D006153:Guanosine Diphosphate; C036852:magnesium GTP; D006160:Guanosine Triphosphate; D018631:ras Proteins