Dataset Information

Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects.

ABSTRACT: Ras-catalyzed guanosine 5' triphosphate (GTP) hydrolysis proceeds through a loose transition state as suggested in our previous study of (18)O kinetic isotope effects (KIE) [ Du , X. et al. ( 2004 ) Proc. Natl. Acad. Sci. U.S.A. 101 , 8858 - 8863 ]. To probe the mechanisms of GTPase activation protein (GAP)-facilitated GTP hydrolysis reactions, we measured the (18)O KIEs in GTP hydrolysis catalyzed by Ras in the presence of GAP(334) or NF1(333), the catalytic fragment of p120GAP or NF1. The KIEs in the leaving group oxygens (the beta nonbridge and the beta-gamma bridge oxygens) reveal that chemistry is rate-limiting in GAP(334)-facilitated GTP hydrolysis but only partially rate-limiting in the NF1(333)-facilitated GTP hydrolysis reaction. The KIEs in the gamma nonbridge oxygens and the leaving group oxygens reveal that the GAP(334) or NF1(333)-facilitated GTP hydrolysis reaction proceeds through a loose transition state that is similar in nature to the transition state of the GTP hydrolysis catalyzed by Ras alone. However, the KIEs in the pro-S beta, pro-R beta, and beta-gamma oxygens suggest that charge increase on the beta-gamma bridge oxygen is more prominent in the transition states of GAP(334)- and NF1(333)-facilitated reactions than that catalyzed by the intrinsic GTPase activity of Ras. The charge distribution on the two beta nonbridge oxygens is also very asymmetric. The catalytic roles of active site residues were inferred from the effect of mutations on the reaction rate and KIEs. Our results suggest that the arginine finger of GAP and amide protons in the P-loop of Ras stabilize the negative charge on the beta-gamma bridge oxygen and the pro-S beta nonbridge oxygen of a loose transition state, whereas Lys-16 of Ras and Mg(2+) are only involved in substrate binding.

SUBMITTER: Du X

PROVIDER: S-EPMC3862183 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects.

Du Xinlin X Sprang Stephen R SR

Biochemistry 20090601 21

Ras-catalyzed guanosine 5' triphosphate (GTP) hydrolysis proceeds through a loose transition state as suggested in our previous study of (18)O kinetic isotope effects (KIE) [ Du , X. et al. ( 2004 ) Proc. Natl. Acad. Sci. U.S.A. 101 , 8858 - 8863 ]. To probe the mechanisms of GTPase activation protein (GAP)-facilitated GTP hydrolysis reactions, we measured the (18)O KIEs in GTP hydrolysis catalyzed by Ras in the presence of GAP(334) or NF1(333), the catalytic fragment of p120GAP or NF1. The KIEs ...[more]

PMID: 19610677

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Project description:Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

Dataset Information

Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects.

Publications

Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects.

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