Dataset Information

Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.

ABSTRACT: Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.

SUBMITTER: Biler M

PROVIDER: S-EPMC7735706 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.

Biler Michal M Crean Rory M RM Schweiger Anna K AK Kourist Robert R Kamerlin Shina Caroline Lynn SCL

Journal of the American Chemical Society 20201112 47

Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metady ...[more]

PMID: 33180505

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Project description:Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the ³¹P chemical shift of bound P(i). The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ?10?-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P(i) binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.

Project description:Kinetic parameters kex (s-1) and kex/Kd (M-1 s-1) are reported for exchange for deuterium in D2O of the C-6 hydrogen of 5-fluororotidine 5'-monophosphate (FUMP) catalyzed by the Q215A, Y217F, and Q215A/Y217F variants of yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 8.1, and by the Q215A variant at pD 7.1-9.3. The pD rate profiles for wildtype ScOMPDC and the Q215A variant are identical, except for a 2.5 log unit downward displacement in the profile for the Q215A variant. The Q215A, Y217F and Q215A/Y217F substitutions cause 1.3-2.0 kcal/mol larger increases in the activation barrier for wildtype ScOMPDC-catalyzed deuterium exchange compared with decarboxylation, because of the stronger apparent side chain interaction with the transition state for the deuterium exchange reaction. The stabilization of the transition state for the OMPDC-catalyzed deuterium exchange reaction of FUMP is ca. 19 kcal/mol smaller than the transition state for decarboxylation of OMP, and ca. 8 kcal/mol smaller than for OMPDC-catalyzed deprotonation of FUMP to form the vinyl carbanion intermediate common to OMPDC-catalyzed reactions OMP/FOMP and UMP/FUMP. We propose that ScOMPDC shows similar stabilizing interactions with the common portions of decarboxylation and deprotonation transition states that lead to formation of this vinyl carbanion intermediate, and that there is a large ca. (19-8) = 11 kcal/mol stabilization of the former transition state from interactions with the nascent CO2 of product. The effects of Q215A and Y217F substitutions on kcat/Km for decarboxylation of OMP are expressed mainly as an increase in Km for the reactions catalyzed by the variant enzymes, while the effects on kex/Kd for deuterium exchange are expressed mainly as an increase in kex. This shows that the Q215 and Y217 side chains stabilize the Michaelis complex to OMP for the decarboxylation reaction, compared with the complex to FUMP for the deuterium exchange reaction. These results provide strong support for the conclusion that interactions which stabilize the transition state for ScOMPDC-catalyzed decarboxylation at a nonpolar enzyme active site dominate over interactions that destabilize the ground-state Michaelis complex.

Dataset Information

Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.

Publications

Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.

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