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Papers by David Alejandro Garzon Barrios

Research paper thumbnail of The Important Role of Covalent Anchor Positions in Tuning Catalytic Properties of a Rationally Designed MnSalen-Containing Metalloenzyme

ACS Catalysis, 2011

Two questions important to the success in metalloenzyme design are how to attach or anchor metal ... more Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta-and para-substituted substrates. The 1•Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min-1) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Research paper thumbnail of Noncovalent Modulation of pH-Dependent Reactivity of a Mn-Salen Cofactor in Myoglobin with Hydrogen Peroxide

Chemistry - A European Journal, 2009

To demonstrate protein modulation of metal cofactor reactivity through non-covalent interactions,... more To demonstrate protein modulation of metal cofactor reactivity through non-covalent interactions, pH-dependent sulfoxidation and ABTS oxidation reactivity of a designed myoglobin (Mb) containing non-native MnSalen complex (1) was investigated using H 2 O 2 as the oxidant. Incorporation of 1 inside the Mb resulted in increase in turnover numbers through exclusion of water from the metal complex and prevention of MnSalen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen bonding interactions resulted in no increase in turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen bond interactions resulted in increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the MnSalen complex. Nonlinear least-squares fitting of rate vs. pH plots demonstrates that 1•Mb(H64X, X= H, R and F) and the control MnSalen 1 exhibit pKas varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1•Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1•Mb displays another pKa at low pH, with pKa of 5.0±0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen bonding interactions by the distal ligand to bind, orient and activate H 2 O 2 is very important for designing artificial catalysts with dramatic different and tunable reactivity from catalysts without proteins.

Research paper thumbnail of The Important Role of Covalent Anchor Positions in Tuning Catalytic Properties of a Rationally Designed MnSalen-Containing Metalloenzyme

ACS Catalysis, 2011

Two questions important to the success in metalloenzyme design are how to attach or anchor metal ... more Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta-and para-substituted substrates. The 1•Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min-1) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Research paper thumbnail of Noncovalent Modulation of pH-Dependent Reactivity of a Mn-Salen Cofactor in Myoglobin with Hydrogen Peroxide

Chemistry - A European Journal, 2009

To demonstrate protein modulation of metal cofactor reactivity through non-covalent interactions,... more To demonstrate protein modulation of metal cofactor reactivity through non-covalent interactions, pH-dependent sulfoxidation and ABTS oxidation reactivity of a designed myoglobin (Mb) containing non-native MnSalen complex (1) was investigated using H 2 O 2 as the oxidant. Incorporation of 1 inside the Mb resulted in increase in turnover numbers through exclusion of water from the metal complex and prevention of MnSalen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen bonding interactions resulted in no increase in turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen bond interactions resulted in increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the MnSalen complex. Nonlinear least-squares fitting of rate vs. pH plots demonstrates that 1•Mb(H64X, X= H, R and F) and the control MnSalen 1 exhibit pKas varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1•Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1•Mb displays another pKa at low pH, with pKa of 5.0±0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen bonding interactions by the distal ligand to bind, orient and activate H 2 O 2 is very important for designing artificial catalysts with dramatic different and tunable reactivity from catalysts without proteins.