Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study.
The active GTP-bound form of p21ras is converted to the biologically inactive GDP-bound form by enzymatic hydrolysis and this function serves to regulate the wild-type ras protein. The side chain of the amino acid at position 61 may play a key role in this hydrolysis of GTP by p21. Experimental studies that define properties of the Q61E mutant of p21H-ras are presented along with supporting molecular dynamics simulations. We find that under saturating concentrations of GTP the Q61E mutant of p21H-ras has a 20-fold greater rate of intrinsic hydrolysis (kcat = 0.57 min-1) than the wild type. The affinity of the Q61E variant for GTP (Kd = 115 microM) is much lower than that of the wild type. GTPase activating protein does not activate the variant. From molecular dynamics simulations, we find that both the wild type and Q61E mutant have the residue 61 side chain in transient contact with a water molecule that is well-positioned for hydrolytic attack on the gamma phosphate. Thr-35 also is found to form a transient hydrogen bond with this critical water. These elements may define the catalytic complex for hydrolysis of the GTP [Pai et al. (1990) EMBO J. 9, 2351]. Similarly, the G12P mutant, which also has an intrinsic hydrolysis rate similar to the wild type, is found to form the same complex in simulation. In contrast, molecular dynamics analysis of the mutants G12R, G12V, and Q61L, which have much lower intrinsic rates than the wild-type p21, do not show this complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Frech
M
M
Institute de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France.
Darden
T A
TA
Pedersen
L G
LG
Foley
C K
CK
Charifson
P S
PS
Anderson
M W
MW
Wittinghofer
A
A
D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005973:Glutamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D005973:Glutamine; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)