Deuterium Isotope Effect in γ‐Aminobutyric Acid Transamination: Determination of Rate‐Limiting Step

Yu, Peter H.; Durden, David A.; Davis, Bruce A.; Boulton, Alan A.

doi:10.1111/j.1471-4159.1987.tb04112.x

Cited by 8 publications

(5 citation statements)

References 17 publications

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“…Despite having K i values that suggest similar active-site binding affinity, all compounds exhibited markedly low substrate activity when compared with GABA. It was hypothesized that their rigid structure may require a binding conformation that orients the requisite proton unfavorably relative to the active-site base responsible for deprotonation, the rate-determining step for turnover of GABA, 40 and therefore decreasing turnover rates.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

Hawker

Silverman

2012

Bioorganic & Medicinal Chemistry

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Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely, γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and L-glutamic acid, an excitatory neurotransmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological diseases. Because of GABA’s poor ability to cross the blood-brain barrier (BBB), a successful strategy to raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inactivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be used as the basis for the design of novel enzyme inactivators.

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Section: Resultsmentioning

confidence: 99%

Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

Hawker

Silverman

2012

Bioorganic & Medicinal Chemistry

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“…Although the binding energies are lower, the rate constants for (−)- 4 and d,l - 6 are about 4 times higher than that for (+)- 3 . Since the rate-determining step is γ-proton removal, removal of this proton in (+)- 3 must be a lower energy process than in the case of GABA, (−)- 4 , or d,l - 6 .…”

Section: Resultsmentioning

confidence: 99%

Inhibition and Substrate Activity of Conformationally Rigid Vigabatrin Analogues with γ-Aminobutyric Acid Aminotransferase

1999

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Several cyclopentene GABA analogues were synthesized as conformationally rigid analogues of the epilepsy drug vigabatrin and tested as inhibitors and substrates of gamma-aminobutyric acid aminotransferase (GABA-AT). None of these compounds produced time-dependent inhibition. (1R, 4S)-(+)-4-Amino-2-cyclopentene-1-carboxylic acid ((+)-3), (4R)-(-)-4-amino-1-cyclopentene-1-carboxylic acid ((-)-4), and d, l-3-amino-1-cyclopentene-1-carboxylic acid (6) are good substrates. The K(m) and k(cat) values for the latter two compounds are very similar to those of GABA, suggesting that they bind in an orientation similar to that of GABA. The K(m) value for (+)-3 is 24 times lower than that for GABA, although its k(cat) value is only one-fourth that for GABA; nonetheless, it is a better substrate for GABA-AT than is GABA. All of these compounds, as well as the enantiomers of 3 and 4 and d, l-trans-4-amino-2-cyclopentene-1-carboxylic acid (5), are competitive inhibitors of GABA-AT. These results demonstrate the effects of the carboxylate group orientation and the stereochemistry of the amino and carboxylate groups on the substrate activity and inhibitor activity, and this should be important to the future design of inhibitors of GABA-AT.

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“…The simplified expression for k inact (eq ), with the assumptions mentioned above, indicates a clear correlation of the microscopic rate constant of the rate-limiting step with k inact , and the enhancement of the rate-limiting step would be reflected in the k inact value. The deprotonation step is known to be the rate-limiting step for GABA-AT with respect to its substrate GABA, whereas the covalent modification step, which is complementary to the water-mediated nucleophilic attack step (Step 4), is known to be the rate-limiting step for inactivators such as γ-vinyl GABA. , Therefore, in the proposed inactivation mechanism, the rate-limiting step could be either the deprotonation step (Step 2) or the water-mediated nucleophilic attack step (Step 4), given the intramolecular nature of both steps. As we point out below, it is crucial to determine the rate-limiting step of the inactivation mechanism to understand the origin of the efficient inactivation of GABA-AT by OV329 compared to that by CPP-115.…”

Section: Introductionmentioning

confidence: 99%

“…As we point out below, it is crucial to determine the rate-limiting step of the inactivation mechanism to understand the origin of the efficient inactivation of GABA-AT by OV329 compared to that by CPP-115. Quantum mechanical (QM) calculations of active-site models and kinetic isotope effect (KIE) studies have proven to be compelling tools in the study of enzymatic reaction mechanisms as well as in the determination of rate-limiting steps, both theoretically and experimentally. − …”

Section: Introductionmentioning

confidence: 99%

Theoretical and Mechanistic Validation of Global Kinetic Parameters of the Inactivation of GABA Aminotransferase by OV329 and CPP-115

2021

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((S)-3-Amino-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (OV329) is a recently discovered inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), which has 10 times better inactivation efficiency than its predecessor, CPP-115, despite the only structural difference being an endocyclic double bond in OV329. Both compounds are mechanism-based enzyme inactivators (MBEIs), which inactivate GABA-AT by a similar mechanism. Here, a combination of a variety of computational chemistry tools and experimental methods, including quantum mechanical (QM) calculations, molecular dynamic simulations, progress curve analysis, and deuterium kinetic isotope effect (KIE) experiments, are utilized to comprehensively study the mechanism of inactivation of GABA-AT by CPP-115 and OV329 and account for their experimentally obtained global kinetic parameters k inact and K I. Our first key finding is that the rate-limiting step of the inactivation mechanism is the deprotonation step, and according to QM calculations and the KIE experiments, k inact accurately represents the enhancement of the rate-limiting step for the given mechanism. Second, the present study shows that the widely used simple QM models do not accurately represent the geometric criteria that are present in the enzyme for the deprotonation step. In contrast, QM cluster models successfully represent both the ground state destabilization and the transition state stabilization, as revealed by natural bond orbital analysis. Furthermore, the globally derived K I values for both of the inactivators represent the inhibitor constants for the initial binding complexes (K d) and indicate the inactivator competition with the substrate according to progress curve analysis and the observed binding isotope effect. The configurational entropy loss accounts for the difference in K I values between the inactivators. The approach we describe in this work can be employed to determine the validity of globally derived parameters in the process of MBEI optimization for given inactivation mechanisms.

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Deuterium Isotope Effect in γ‐Aminobutyric Acid Transamination: Determination of Rate‐Limiting Step

Cited by 8 publications

References 17 publications

Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

Inhibition and Substrate Activity of Conformationally Rigid Vigabatrin Analogues with γ-Aminobutyric Acid Aminotransferase

Theoretical and Mechanistic Validation of Global Kinetic Parameters of the Inactivation of GABA Aminotransferase by OV329 and CPP-115

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