Transition State Analysis of NAD+ Hydrolysis by the Cholera Toxin Catalytic Subunit
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- 作者：Kathleen A. Rising and Vern L. Schramm
- 刊名：Journal of the American Chemical Society
- 出版年：1997
- 出版时间：January 8, 1997
- 年：1997
- 卷：119
- 期：1
- 页码：27 - 37
- 全文大小：282K
- 年卷期：v.119,no.1(January 8, 1997)
- ISSN：1520-5126
文摘
The transition state for NAD+ (oxidized nicotinamideadenine dinucleotide) hydrolysis by the choleratoxin A1 polypeptide (CTA) has been characterized by multiple V/Kkinetic isotope effects (KIEs) using labeledNAD+ as the substrate. CTA causes cholera bycatalyzing the ADP-ribosylation of the signal-transducingGs
protein.In vitro, CTA catalyzes the ADP-ribosylation of severalsimple guanidino compounds as well as the slow hydrolysisof NAD+ (kcat = 8min-1, Km = 14 mM)to form ADP-ribose and nicotinamide. KIEs for NAD+hydrolysis arethe following: primary 14C = 1.030 ± 0.005, primary15N = 1.029 ± 0.004, -secondary 3H =1.186 ± 0.004,
-secondary 3H = 1.108 ± 0.004, 
-secondary3H = 0.986 ± 0.003, 
-secondary 3H =1.020 ± 0.003, and primarydouble = 1.052 ± 0.004. On the basis of steady-state kineticparameters for CTA-catalyzed NAD+ hydrolysis,aswell as a comparison with KIEs measured for NAD+solvolysis, the enzymatic KIEs are near-intrinsic anddescribea transition state that is relatively desolvated at the reactioncenter. The inability of CTA to catalyzeNAD+methanolysis is also consistent with desolvation at the reactioncenter. Together with the observation that CTAcatalyzes ADP-ribosylation with inversion of configuration at theanomeric carbon (Oppenheimer, N. J. J. Biol.Chem. 1978, 253, 4907-4910),NAD+ hydrolysis by CTA is best described by a concerteddisplacement mechanisminvolving an enzyme-directed water nucleophile. The small, inversesolvent deuterium KIE demonstrates that arate-limiting proton transfer does not characterize the CTA reactioncoordinate. Using bond-energy bond-ordervibrational analysis, the KIEs for NAD+ hydrolysis by CTAhave been used to model a transition state geometry.The model is consistent with a highly dissociative, concertedmechanism, characterized by distances from the anomericcarbon to the leaving group and incoming nucleophile of approximately2.2 and 3.3 Å, respectively. There is significantoxocarbonium ion character and hyperconjugation within the ribose ring.The - and -secondary KIEs are evidencefor enzyme-substrate interactions that are remote from the reactioncenter and are unique to enzymatic stabilizationof the transition state.
protein.In vitro, CTA catalyzes the ADP-ribosylation of severalsimple guanidino compounds as well as the slow hydrolysisof NAD+ (kcat = 8min-1, Km = 14 mM)to form ADP-ribose and nicotinamide. KIEs for NAD+hydrolysis arethe following: primary 14C = 1.030 ± 0.005, primary15N = 1.029 ± 0.004, -secondary 3H =1.186 ± 0.004,-secondary 3H = 1.108 ± 0.004, -secondary3H = 0.986 ± 0.003, -secondary 3H =1.020 ± 0.003, and primarydouble = 1.052 ± 0.004. On the basis of steady-state kineticparameters for CTA-catalyzed NAD+ hydrolysis,aswell as a comparison with KIEs measured for NAD+solvolysis, the enzymatic KIEs are near-intrinsic anddescribea transition state that is relatively desolvated at the reactioncenter. The inability of CTA to catalyzeNAD+methanolysis is also consistent with desolvation at the reactioncenter. Together with the observation that CTAcatalyzes ADP-ribosylation with inversion of configuration at theanomeric carbon (Oppenheimer, N. J. J. Biol.Chem. 1978, 253, 4907-4910),NAD+ hydrolysis by CTA is best described by a concerteddisplacement mechanisminvolving an enzyme-directed water nucleophile. The small, inversesolvent deuterium KIE demonstrates that arate-limiting proton transfer does not characterize the CTA reactioncoordinate. Using bond-energy bond-ordervibrational analysis, the KIEs for NAD+ hydrolysis by CTAhave been used to model a transition state geometry.The model is consistent with a highly dissociative, concertedmechanism, characterized by distances from the anomericcarbon to the leaving group and incoming nucleophile of approximately2.2 and 3.3 Å, respectively. There is significantoxocarbonium ion character and hyperconjugation within the ribose ring.The - and -secondary KIEs are evidencefor enzyme-substrate interactions that are remote from the reactioncenter and are unique to enzymatic stabilizationof the transition state.