The transition state for NAD+ (oxidized nicotinamide adenine dinucleotide) hydrolysis by the cholera toxin A1 polypeptide (CTA) has been characterized by multiple V/K kinetic isotope effects (KIEs) using labeled NAD+ as the substrate. CTA causes cholera by catalyzing the ADP-ribosylation of the signal-transducing Gs α protein. In vitro, CTA catalyzes the ADP-ribosylation of several simple guanidino compounds as well as the slow hydrolysis of NAD+ (k cat = 8 min-1, K m = 14 mM) to form ADP−ribose and nicotinamide. KIEs for NAD+ hydrolysis are the following:  primary 14C = 1.030 ± 0.005, primary 15N = 1.029 ± 0.004, α-secondary 3H = 1.186 ± 0.004, β-secondary 3H = 1.108 ± 0.004, γ-secondary 3H = 0.986 ± 0.003, δ-secondary 3H = 1.020 ± 0.003, and primary double = 1.052 ± 0.004. On the basis of steady-state kinetic parameters for CTA-catalyzed NAD+ hydrolysis, as well as a comparison with KIEs measured for NAD+ solvolysis, the enzymatic KIEs are near-intrinsic and describe a transition state that is relatively desolvated at the reaction center. The inability of CTA to catalyze NAD+ methanolysis is also consistent with desolvation at the reaction center. Together with the observation that CTA catalyzes ADP-ribosylation with inversion of configuration at the anomeric carbon (Oppenheimer, N. J. J. Biol. Chem. 1978, 253, 4907−4910), NAD+ hydrolysis by CTA is best described by a concerted displacement mechanism involving an enzyme-directed water nucleophile. The small, inverse solvent deuterium KIE demonstrates that a rate-limiting proton transfer does not characterize the CTA reaction coordinate. Using bond-energy bond-order vibrational analysis, the KIEs for NAD+ hydrolysis by CTA have been used to model a transition state geometry. The model is consistent with a highly dissociative, concerted mechanism, characterized by distances from the anomeric carbon to the leaving group and incoming nucleophile of approximately 2.2 and 3.3 Å, respectively. There is significant oxocarbonium ion character and hyperconjugation within the ribose ring. The γ- and δ-secondary KIEs are evidence for enzyme−substrate interactions that are remote from the reaction center and are unique to enzymatic stabilization of the transition state.

Sesquiterpene synthases are a family of enzymes that catalyze farnesyl pyrophosphate (FPP) cyclization via alternative pathways to produce a variety of cyclic sesquiterpene products. Catalysis by several of these enzymes, including tobacco 5-epi-aristolochene synthase (TEAS), has been proposed to include the formation of germacrene A as a stable intermediate. Neither germacrene A nor any other intermediate is released from sesquiterpene synthase active sites during normal catalysis. Evidence to support the intermediacy of germacrene A has been derived from investigations of aristolochene synthases from Aspergillus terreus and Penicillium roquefortii (Cane, D. E. Chem. Rev. 1990, 90, 1089−1103 and references therein. Cane, D. E.; Bryant, C. J. Am. Chem. Soc. 1994, 116, 12063−12064. Cane, D. E.; Tsantrizos, Y. S. J. Am. Chem. Soc. 1996, 118, 10037−10040). However, until the present investigations of TEAS, formation of this postulated intermediate has never been directly demonstrated. TEAS catalyzes the cyclization of FPP to 5-epi-aristolochene, a precursor of a tobacco phytoalexin, capsidiol. Based upon the three-dimensional structure of TEAS, a detailed mechanism has been proposed for TEAS catalysis that includes the prediction that proton donation by Y520 is responsible for the activation of germacrene A to a eudesmane cation (Starks, C. M.; Back, K.; Chappell, J.; Noel, J. P. Science 1997, 277, 1815−1820). In the present investigation, a Y520F point mutation is introduced into TEAS (TEAS-Y520F) by site-directed mutagenesis. In the presence of 3H−FPP, TEAS-Y520F produces hexanes−extractable 3H with a catalytic efficiency approximately 3% that of nonmutated, recombinant TEAS. The hexanes−extractable 3H is identified as germacrene A, m/z 204, through direct GC-MS comparison to an authentic sample. This observation confirms the intermediacy of germacrene A in TEAS catalysis, supports the postulated production of germacrene A by a variety of other sesquiterpene synthases, and also confirms the proposed role of Y520 in TEAS catalysis.

An enzymatic synthesis is described for the production of NAD+ labeled with a radioactive or stable isotope at any desired position in the AMP or NMN+ portions of the molecule. In the first step, ten enzyme-catalyzed reactions are coupled for the synthesis of nicotinic acid adenine dinucleotide (NaAD+) from glucose, nicotinic acid, and ATP. NAD+ is formed from NaAD+ and glutamine in the second step. Oxidized nicotinamide adenine dinucleotide was synthesized with 3H, l%, or I5N label specifically incorporated in the ribose or nicotinamide of the NMN+ portion of NAD+ as [HN~'-~HH]NAD+, [ H N~' -~H ] N A D + , [HN~'-~H]NAD+, [HN~'-~HH]NAD+, [CN~'-~%]NAD+, [cN5'-WC]NAD+, ["1-I5N, CN~'-'%]NAD+, and ["1-l5N, CNS'-'%]NAD+. Nuclear magnetic resonance spectroscopy of [ H N~' -~H ] N A D +as well as enzymatic degradation were used to verify the position of labels. Appropriately labeled glucose, ribose 5-phosphate, or nicotinic acid were the starting materials and were converted to NAD+ using enzymes from the pentose pathway and the pathway for NAD+ de nouo synthesis. Yields of purified NAD+ to 96% were obtained from starting glume. The labeled NAD+ is catalytically competent and is chromatographically and spectrophotometrically indistinguishable from authentic NAD+. By using specifically labeled ATP as a precursor (Parkin, D. W.; Schramm, V. L. Biochemistry 1987,26,913-920), the method is readily adaptable for the synthesis of NAD+ with single or multiple atomic labels at various positions in the AMP portion of the molecule. NAD+ was synthesized from [8-W]ATP to give [C&W]NAD+ as an example. Together these methods provide a general scheme for the efficient synthesis of NAD+ of high purity with 3H. 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule.