scholarly journals The Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

2021 ◽  
Author(s):  
Martin Pfeiffer ◽  
Bernd Nidetzky ◽  
Rory Crean ◽  
Cátia Moreira ◽  
Antonietta Parracino ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Phosphoryl Transfer ◽  
Enzyme Design ◽  
Linear Free Energy ◽  
Mechanistic Pathway ◽  
Empirical Valence Bond ◽  
Energy Relationships

Cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. A deepened understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes, and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for cooperative interplay between the nucleophilic and general-acid catalytic groups in the wildtype enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is around 10^4-fold. Cooperativity with the catalytic acid adds ≥10^2-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

scholarly journals Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

2020 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Computational Procedure ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Stability Ranking ◽  
Random Library

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

2020 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Computational Procedure ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Stability Ranking ◽  
Random Library

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

2020 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Computational Procedure ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Stability Ranking ◽  
Random Library

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

Linear Free Energy Relationships for N(7)-Substituted Guanosines as Substrates of Calf Spleen Purine Nucleoside Phosphorylase. Possible Role of N(7)-Protonation as an Intermediary in Phosphorolysis

1993 ◽  
Vol 48 (9-10) ◽  
pp. 803-811 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Ewa Kulikowska ◽  
David Shugar
Keyword(s):  
Acid Hydrolysis ◽  
Purine Nucleoside Phosphorylase ◽  
Catalytic Mechanism ◽  
Enzymatic Reaction ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Linear Free Energy ◽  
Repair Enzymes ◽  
Substrate Properties ◽  
Energy Relationships

Abstract Quantitative structure-activity relationships (QSAR) for a series of N(7)-substituted guanosines as substrates for calf spleen purine nucleoside phosphorylase (PNP) were developed, and compared with those for acid hydrolysis of these analogues. There is no correlation between the rates for enzymatic phosphorolysis and acid hydrolysis, indicating that for the enzymatic reaction labilization of the glycosidic bond is not the only, nor the predominant, effect of N(7)-substitution. Multiple regression analysis of the enzymatic process revealed that optimal substrate properties (minimal Michaelis constant) are associated with the Taft electronic constant equal zero and a substituent size, parametrized by the Taft steric constant, smaller than that for a methyl group. These results support the hypothesis of protonation of the N(7)-position of the base by the enzyme as a catalytic mechanism for calf spleen PNP. Attention is drawn to the postulated similar mechanism of action of other purine N-glycosidases, including plant antiviral proteins which function as RNAN-glycosidases, and possibly some DNA N-glycosidases which function as repair enzymes

scholarly journals Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis byPseudomonas aeruginosaArylsulfatase

2018 ◽  
Author(s):  
Bert van Loo ◽  
Ryan Berry ◽  
Usa Boonyuen ◽  
Mark F. Mohamed ◽  
Marko Golicnik ◽  
...  
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
Charge Compensation ◽  
Linear Free Energy ◽  
Uncatalyzed Reaction ◽  
Leaving Group ◽  
Energy Relationships ◽  
Increased Sensitivity ◽  
Leaving Group Ability ◽  
Active Site Mutants

ABSTRACTPseudomonas aeruginosaarylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleavingroupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211.15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S-Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

scholarly journals Laser Photolysis Investigations of Ligand Binding With Models of the Active Site of Respiratory Hemoproteins: Kinetic and Thermodynamic Aspects

1990 ◽  
Vol 10 (5-6) ◽  
pp. 297-318 ◽  
Author(s):  
D. Lavalette ◽  
C. Tetreau ◽  
M. Momenteau ◽  
J. Mispelter ◽  
J. M. Lhoste ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Parameters ◽  
Dissociation Rate ◽  
Laser Photolysis ◽  
Concentration Jump ◽  
Linear Free Energy ◽  
Energy Relationships ◽  
Dissociation Rate Constants ◽  
Kinetics Of

During the past 15 years, laser photolysis has been the method of choice for probing the complex reaction kinetics of respiratory proteins. In an attempt to determine the structural parameters which govern their reactivity, synthetic heme model compounds capable of simulating particular aspects of the reactivity of the active site of hemoproteins have been successively proposed. Laser photolysis of heme compounds merely induces a reversible photodissociation of one ligand at a time. This is equivalent to performing a fast concentration jump "in situ" and provides a powerful, fast and "clean" chemical relaxation technique. To gather association and dissociation rate constants of various ligands (O2, CO, nitrogenous bases) special methods have been developed or adapted. The problem of comparing and classifying a large number of collected data has been greatly simplified by introducing a Linear Free Energy Relationships formalism. In the first part of this paper, some of the methods and concepts which have emerged from several years of investigations of heme proteins and heme models and which are of a sufficient generality to be useful in other fields of chemical kinetics are reviewed. In the second part of the paper we present the application of the preceding methods to a kinetic study of a series of heme models which were specifically designed to investigate the important problem of H-bonding as a stabilizing factor of the oxygenated heme model and hemoprotein complexes.

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