Altering the binding determinant on the interdigitating loop of mandelate racemase shifts specificity towards that of d-tartrate dehydratase

Nagar, Mitesh; Hayden, Joshua A.; Sagey, Einat; Worthen, George; Park, Mika; Sharma, Apurva; Fetter, Christopher M.; Kuehm, Oliver P.; Bearne, Stephen L.

doi:10.1016/j.abb.2022.109119

Cited by 3 publications

(2 citation statements)

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“…Although the kinetics of some of these MR variants had been described previously by our lab for the fusion proteins with an N-terminal His 6 -tag, better yields are obtained using the StrepII-tagged proteins . These variants, along with seven additional variants that were reported previously (L93K, L93D, L93R, Y137F, Y137L, K164R, and K166R), were selected because the substituted amino acids were at the active site and because the variant enzymes possessed sufficient catalytic activity to permit inhibition studies. Overall, the kinetic parameters ( K m , k cat , and k cat / K m ) for wild-type MR and the 11 MR variants were determined along with the corresponding competitive inhibition constants ( K i ) accompanying binding of 3,4-diCl-PBA (Table S4 and Figures S19–S30).…”

Section: Resultsmentioning

confidence: 99%

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A Phenylboronic Acid-Based Transition State Analogue Yields Nanomolar Inhibition of Mandelate Racemase

2023

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Mandelate racemase (MR) catalyzes the Mg2+-dependent interconversion of (R)- and (S)-mandelate by stabilizing the altered substrate in the transition state (TS) by ∼26 kcal/mol. The enzyme has been employed as a model to explore the limits to which the free energy of TS stabilization may be captured by TS analogues to effect strong binding. Herein, we determined the thermodynamic parameters accompanying binding of a series of bromo-, chloro-, and fluoro-substituted phenylboronic acids (PBAs) by MR and found that binding was predominately driven by favorable entropy changes. 3,4-Dichloro-PBA was discovered to be the most potent inhibitor yet identified for MR, binding with a K d app value of 11 ± 2 nM and exceeding the binding of the substrate by ∼72,000-fold. The ΔC p value accompanying binding (−488 ± 18 cal·mol–1 K–1) suggested that dispersion forces contribute significantly to the binding. The pH-dependence of the inhibition revealed that MR preferentially binds the anionic, tetrahedral form of 3,4-dichloro-PBA with a pH-independent K i value of 5.7 ± 0.5 nM, which was consistent with the observed upfield shift of the 11B NMR signal. The linear free energy relationship between log(k cat/K m) and log(1/K i) for wild-type and 11 MR variants binding 3,4-dichloro-PBA had a slope of 0.8 ± 0.2, indicating that MR recognizes the inhibitor as an analogue of the TS. Hence, halogen substitution may be utilized to capture additional free energy of TS stabilization arising from dispersion forces to enhance the binding of boronic acid inhibitors by MR.

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Section: Resultsmentioning

confidence: 99%

“…Each mutant open reading frame was sequenced using commercial automated DNA sequencing (Robarts Research Institute, London, ON, Canada) to ensure that no other alterations in the nucleotide sequence had been introduced. All other variants were prepared using previously described protocols. ,,− …”

Section: Methodsmentioning

confidence: 99%