Transition State Analysis of NAD⁺Hydrolysis by the Cholera Toxin Catalytic Subunit

Abstract

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.

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

“…Data from our laboratory and others support the critical role played by the active-site-occluding loop in the activity of the (39). In LT, substitutions within the active-site-occluding loop of residues that are found in the related LT-II toxins also affect structure and function (8).…”

Enzymatic Transition States and Transition State Analog Design

“…The results (Figure 16) indicate that the transition states for hydrolysis are similar for pH-independent hydrolysis (pH values from 3 to 7) and for the slow NAD + hydrolysis reactions catalyzed by all three enzymes when acceptor proteins are not present (107)(108)(109)(110). In every case the ribosyl group has well-developed oxocarbenium ion character, with nearly full positive charge at the transition state.…”

Transition State Structure for the Hydrolysis of NAD+Catalyzed by Diphtheria Toxin

“…All the 3 H KIEs were measured using 5′-14 C as the remote label, which was shown to have no measurable KIE for cholera toxin or solvolytic hydrolysis of NAD +. 28 The 4′- 18 …”