PH Dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42.
ABSTRACT: An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H side chain at high pH, the enzyme retains lyase activity at ?100-fold lowered catalytic efficiency but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid side chains.
Project description:The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non-native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.
Project description:PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5-2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or 2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic side chain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267-0.505 kcal/mol of catalytic free energy, or roughly 6-11% rescue).
Project description:For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.
Project description:The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.
Project description:A study of the Thermus thermophilus chorismate mutase (TtCM) is described by using quantum mechanics (self-consistent-charge density-functional tight binding)/molecular mechanics, umbrella sampling, and the weighted histogram analysis method. The computed free energies of activation for the reactions in water and TtCM are comparable to the experimental values. The free energies for formation of near attack conformer have been determined to be 8.06 and 0.05 kcal/mol in water and TtCM, respectively. The near attack conformer stabilization contributes approximately 90% to the proficiency of the enzymatic reaction compared with the reaction in water. The transition state (TS) structures and partial atom charges are much the same in the enzymatic and water reactions. The difference in the electrostatic interactions of Arg-89 with O13 in the enzyme-substrate complex and enzyme-TS complex provides the latter with but 0.55 kcal/mol of 1.92 kcal/mol total TS stabilization. Differences in electrostatic interactions between components at the active site in the enzyme-substrate complex and enzyme-TS complex are barely significant, such that TS stabilization is of minor importance and the enzymatic catalysis is through an entropic advantage.
Project description:The hemoglobin of Synechococcus sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When b heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent pKa of 9.3 and a Lys-ligated population of ?60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20-30 s-1 by NMR exchange spectroscopy. Lys42-His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome c, in which Lys-Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only.
Project description:Here, we present the crystal structure of the ecdysone phosphate phosphatase (EPPase) phosphoglycerate mutase (PGM) homology domain, the first structure of a steroid phosphate phosphatase. The structure reveals an alpha/beta-fold common to members of the two histidine (2H)-phosphatase superfamily with strong homology to the Suppressor of T-cell receptor signaling-1 (Sts-1 PGM) protein. The putative EPPase PGM active site contains signature residues shared by 2H-phosphatase enzymes, including a conserved histidine (His80) that acts as a nucleophile during catalysis. The physiological substrate ecdysone 22-phosphate was modeled in a hydrophobic cavity close to the phosphate-binding site. EPPase PGM shows limited substrate specificity with an ability to hydrolyze steroid phosphates, the phospho-tyrosine (pTyr) substrate analogue para-nitrophenylphosphate ( pNPP) and pTyr-containing peptides and proteins. Altogether, our data demonstrate a new protein tyrosine phosphatase (PTP) activity for EPPase. They suggest that EPPase and its closest homologues can be grouped into a distinct subfamily in the large 2H-phosphatase superfamily of proteins.
Project description:The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.
Project description:Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min-1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min-1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the corresponding location of Candida lipases is uncharged due to other active site differences and does not cause the same distortion. This example of the fine tuning of the same catalytic triad for different types of catalysis by subtle interactions with other active site residues shows how difficult it is to design new catalytic reactions of enzymes.
Project description:The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.