Synthesis of [g ¹⁸O₄]GTP and [g ¹⁸O₃]GTP. [¹⁸O₄]P_i was synthesized as the tetrabutylammonium salt from PCl₅ and H₂¹⁸O (97% enriched; Isotech) (1). Carbamate kinase (Sigma) was used to catalyze the formation of [g ¹⁸O₃]GTP from [¹³C]GDP and carbamoyl [ ¹⁸O₄]phosphate (2). Activation of GDP by carbonyldiimidazole allowed the addition of [¹⁸O₄]P_i to generate [g ¹⁸O₄]GTP (3).

Synthesis of [b ¹⁸O₃]GTP and [b ¹⁸O₂]GTP. [b ¹⁸O₃]GDP is a common intermediate for the synthesis of both [b ¹⁸O₃]GTP and [b ¹⁸O₂]GTP. [b ¹⁸O₃, b -g ¹⁸O, g ¹⁸O₃]GTP was first synthesized in a coupled reaction catalyzed by carbamate kinase and guanylate kinase, then hydrolyzed by Ga _i1 to yield [b ¹⁸O₃]GDP, as follows. Carbamoyl [¹⁸O₄]phosphate was synthesized by incubating a reaction mixture containing 180 m l [ ¹⁸O₄]P_i (0.6 M), 170 m l KCNO (3 M), and 340 m l sodium acetate (1M, pH 4.9) at 37°C for 20 min. To this mixture was added 30 mg of GMP, 1 mg of [b ¹⁸O₃]GDP (or, if not available, 0.3 mg of natural abundance ADP), 200 m l of Hepes (500 mM, pH 7.5), 40 m l of MgCl₂ (1 M), 9 units of guanylate kinase, and 50 units of carbamate kinase and the reaction mixture was incubated overnight. The [b ¹⁸O₃]GDP (or ADP) in the reaction mixture serves as both acceptor and donor of [¹⁸O₄]P_i , as it receives [¹⁸O₄]P_i in the reaction catalyzed by carbamate kinase and then donates the acquired [¹⁸O₄]P_i to GMP in the reaction catalyzed by guanylate kinase to generate more [b ¹⁸O₃]GDP. The newly generated [b ¹⁸O₃]GDP goes through the same cycle until all nucleotide is converted to [b ¹⁸O₃, b -g ¹⁸O, g ¹⁸O₃]GTP, provided that [¹⁸O₄]P_i is present in excess. [b ¹⁸O₃, b -g ¹⁸O, g ¹⁸O₃]GTP was then purified by anion exchange chromatography and incubated with 1 mg Ga _i1 in 200 mM ammonium sulfate, 1 mM MgCl₂, 20 mM Tris·HCl, pH7.5. The conversion of [b ¹⁸O₃, b -g ¹⁸O, g ¹⁸O₃]GTP to [b ¹⁸O₃]GDP was complete overnight. Carbamate kinase was then used to couple [b ¹⁸O₃]GDP and carbamoyl phosphate to generate [b ¹⁸O₃]GTP (2). Alternatively, chemical activation by carbonyldiimidazole allowed the coupling of [b ¹⁸O₃]GDP and phosphate to generate [b ¹⁸O₂]GTP (3).

Nucleotides were purified to homogeneity by anion exchange chromatography using 1- or 5-ml Hi-Trap columns. Triethylamonium bicarbonate or ammonium acetate were used as elution buffers and removed from the purified product by lyophilization. Electrospray triple quadrupole mass spectrometry was used to deduce the isotopic purity of ¹³C and ¹⁸O in the labeled nucleotides. ¹³C isotopic purity is >99% for the [¹³C]GMP and [¹³C]GTP purchased from Spectra Gases (Branchburg, NJ). The ¹⁸O isotopic purities for [g ¹⁸O₃]GTP and [b ¹⁸O₃]GTP are 96%. The ¹⁸O isotopic purities for [g ¹⁸O₄]GTP and [b ¹⁸O₂]GTP are ~94%. Two lots of ¹³C-depleted GTP were purchased from Spectra Gases. The residual carbon isotope ratios for the two lots were determined to be 0.182 and 0.321, respectively, by HPLC/chemical reaction interface (CRI)/isotope ration mass spectrometry (IRMS).

Preparation of Ras in complex with [¹⁸O, ¹³C]GTP and [¹²C]GTP. Ras copurifies with bound GDP because of the high affinity of GDP for Ras. It is desirable to remove this endogenous GDP so that the GDP generated from the hydrolysis of the substrate mixture can be used for kinetic isotope effect (KIE) analysis. For this purpose, Ras is first stripped of GDP and reloaded with a caged GTP analog, hydroxyphenacyl GTP (HPA-GTP), that can be readily displaced with isotopically labeled substrate (2) as follows. To 1 ml of a solution containing 10 mg His-tagged Ras, 200 mM ammonium sulfate, and 20 mM Tris·HCl, pH 8.0, was added 1 m l of alkaline phosphatase (Sigma P5521) and 2-fold excess of HPA-GTP. The disappearance of GDP was monitored by using anion exchange column chromatography. After GDP was completely cleaved, the Ras•HPA-GTP complex was purified by Ni²⁺ affinity chromatography to remove alkaline phosphatase. Typically, the yield of Ras•HPA-GTP is 30-40%. The Ras•HPA-GTP complex was then divided into 0.6-mg fractions. To each fraction was added a molar equivalent of isotopically labeled substrate mixture in water and EDTA (1 mM). HPA-GTP was then removed by using an Amicon Ultra concentration device. The Ras complex with isotopically labeled substrates is used immediately for KIE experiments.

1. Lowe, G. & Sproat, B. (1978) J. Chem. Soc. Perkin I , 1622-1630.

2. Du, X., Frei, H. & Kim, S. H. (2000) J. Biol. Chem. 275, 8492-8500.

3. Webb, M. (1980) Biochemistry 19, 4744-4748.