scholarly journals Characterization of the interactions between Asp141 and Phe236 in the Mn2+–l-malate binding of pigeon liver malic enzyme

2003 ◽  
Vol 374 (3) ◽  
pp. 633-637 ◽  
Author(s):  
Yen-I CHEN ◽  
Yu-Hou CHEN ◽  
Wei-Yuan CHOU ◽  
Gu-Gang CHANG
Keyword(s):  
Metal Binding ◽  
Malic Enzyme ◽  
Double Mutant ◽  
Dissociation Constants ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Coupling Energy ◽  
Metal Ligands ◽  
Quaternary Structures ◽  
Pigeon Liver

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+–ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141→Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (ΔΔGint) between any two residues. The dissociation constants for Mn2+ (Kd,Mn) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the Kd,Mn for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of −2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.

scholarly journals Duck liver ‘malic’ enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

1992 ◽  
Vol 284 (3) ◽  
pp. 869-876 ◽  
Author(s):  
R Y Hsu ◽  
M J Glynias ◽  
J Satterlee ◽  
R Feeney ◽  
A R Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Malic Enzyme ◽  
Dissociation Constants ◽  
Substrate Inhibition ◽  
Oxidative Decarboxylation ◽  
Lacz Gene ◽  
Wild Type ◽  
Recombinant Enzymes ◽  
Wild Type Enzyme

A cDNA for duck liver ‘malic’ enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ′ gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka′ (270 microM), dissociation constants of Mn2+ at ‘tight’ (activating) and ‘weak’ metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the ‘weak’ sites (i.e. inhibition by L-malate, Ka′); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Biochemistry ◽  
1994 ◽  
Vol 33 (25) ◽  
pp. 7931-7936 ◽  
Author(s):  
Chien-Hwa Wei ◽  
Wei-Yuan Chou ◽  
Shih-Ming Huang ◽  
Ching-Chun Lin ◽  
Gu-Gang Chang
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Malic Enzyme ◽  
Metal Binding Site ◽  
Affinity Cleavage ◽  
Pigeon Liver

Binding Properties of a Human Anti-Ib ScFv Antibody (HIb-1) to Native, Recombinant, and Transgenicially Expressed Human Platelet GP Ibα Antigen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4162-4162
Author(s):  
Antony Y. Kim ◽  
George Bell ◽  
Joshua Lovelock ◽  
Elizabeth G. Favre ◽  
Tapan K. Biswas ◽  
...  
Keyword(s):  
Double Mutant ◽  
Dextran Sulfate ◽  
Dissociation Constants ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Model Systems ◽  
Acute Injury ◽  
Wild Type ◽  
Single Chain ◽  
Amino Terminal

Abstract HIb-1 is a single chain antibody (ScFv) originally obtained from the Griffin.1 library. ScFv from this library are derived from the in vitro recombination of germline human immunoglobulin VH and VL chain segments whose diversity has been greatly increased by mutagenesis directed at the CDR3 regions. HIb-1 was selected from the library by sequential biopanning first against CHO cells surface-expressing human GPIbα, and then against human platelets. Previous studies using Western blot analysis have shown that HIb-1 binds to an epitope within the 45 kDa amino-terminal portion of GPIbα (Lapan, Lyle, and Miller, 1999, Thromb. Haemost. 82S:393). HIb-1 displays inhibitory activity against both ristocetin-induced and shear-induced platelet aggregation. Biacore surface plasmon resonance analysis was used to establish dissociation constants between HIb-1and either the extracellular domain of GPIbα (i.e., “glycocalicin” or GC) derived from normal human platelets or the CHO-cell secreted GPIbα1–483 recombinant extracellular protein. With native GC immobilized on dextran sulfate, the KD for binding by analyte HIb-1 was ~600nM. With the wild-type recombinant GPIbα1–483 bound to dextran sulfate, the KD was only slightly higher at ~900 nM. We additionally studied a double mutant increase-of-function GPIbα1-483 containing the Gly233→Val233 and Met239→Val239 substitituions. The KD of HIb-1 binding to the double mutant was virtually identical to that of the wild-type, at ~900 nM. Binding of HIb-1 to native GC immobilized in an ELISA assay format was also performed. HIb-1 showed saturable binding, with a half-maximal binding concentration of approximately 170 nM. Flow cytometric analysis was also performed on platelets from murine GPIbα-null (i.e., “Bernard-Soulier”) mice that had been rescued with the human GPIbα transgene (Ware, Russell, and Ruggeri, 2000, PNAS, 97:2803), as well as upon normal mice. Murine platelets were gated using a rat anti-murine GPIIb/IIIa antibody. HIb-1 did not bind to normal murine platelets, but showed strong binding to platelets from the transgenic animals, comparable in intensity to that seen with the GPIbα mab SZ-2 (for which quantitative bead analysis indicated approximately 8,000 GPIbα receptors per platelet). It should be noted that the binding capabilities of HIb-1 to human GPIbα are those of a monovalent ScFv molecule of approximately 30 kDa, and may be sufficient for studies of in vivo GPIbα inhibition either in the case of acute injury model or of longer-term models involving repeated dosing. Model systems of more chronic inhibition, however, may require conversion of the ScFv into IgG molecules, in order to achieve greater avidity.

scholarly journals The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

2009 ◽  
Vol 53 (10) ◽  
pp. 4464-4471 ◽  
Author(s):  
Carine Bebrone ◽  
Heinrich Delbrück ◽  
Michaël B. Kupper ◽  
Philipp Schlömer ◽  
Charlotte Willmann ◽  
...  
Keyword(s):  
Active Site ◽  
Aeromonas Hydrophila ◽  
Zinc Ion ◽  
Amide Bond ◽  
Zinc Ions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
X Ray ◽  
Metal Ligands ◽  
Class B

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

scholarly journals Zinc- and iron-dependent cytosolic metallo-β-lactamase domain proteins exhibit similar zinc-binding affinities, independent of an atypical glutamate at the metal-binding site

2004 ◽  
Vol 385 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Oliver SCHILLING ◽  
Andreas VOGEL ◽  
Brenda KOSTELECKY ◽  
Hugo NATAL da LUZ ◽  
Daniel SPEMANN ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Dissociation Constants ◽  
Zinc Binding ◽  
Titration Calorimetry ◽  
Good Correspondence ◽  
Metal Binding Site ◽  
E Coli ◽  
Metal Ligands ◽  
Metal Selectivity

ZiPD (zinc phosphodiesterase; synonyms are ElaC, ecoZ, RNaseZ and 3′ tRNase) and the iron-dependent redox enzyme FlRd (flavorubredoxin) from Escherichia coli represent prototypical cases of proteins sharing the metallo-β-lactamase fold that require strict metal selectivity for catalytic activity, yet their metal selectivity has only been partially understood. In contrast with hydrolytic metallo-β-lactamase proteins, iron-dependent FlRd-like enzymes have an atypical glutamate ligand, which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-β-lactamase domain is capable of incorporating two zinc ions into the binuclear metal-binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (Kd1=2.2±0.2 μM and Kd2=23.0±0.6 μM) and to the E. coli FlRd metallo-β-lactamase domain (Kd1=0.7±0.1 μM and Kd2=26.0±0.1 μM). In good correspondence, apo-ZiPD requires incubation with 10 μM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an increased first zinc dissociation constant (Kd1=30±4 μM and Kd2=23±2 μM). In combination with a recent mutational study on ZiPD [Vogel, Schilling and Meyer-Klaucke (2004) Biochemistry 43, 10379–10386], we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-β-lactamase domain but suppresses possible hydrolytic cross-activity.

Enhancement of Acid Stability of Alpha Amylase from Bacillus licheniformis by Error-Prone PCR

2013 ◽  
Vol 774-776 ◽  
pp. 664-669
Author(s):  
Yan Jing Xu ◽  
Yi Han Liu ◽  
Shuai Fan ◽  
Fu Ping Lu
Keyword(s):  
Bacillus Licheniformis ◽  
Double Mutant ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Alpha Amylase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Chain Reaction ◽  
Acid Stability ◽  
Polymerase Chain

Acid stability of Bacillus licheniformis alpha amylase (BLA) was improved by error-prone polymerase chain reaction. The mutated BLA gene was expressed in Escherichia coli. An acid stability double mutant (K344R/H405R in BLA) was isolated. Two single mutants K344R and H405R were obtained by the way of site-directed mutagenesis. The enzymes (BLA) of the three mutants were isolated and characterized. Kinetic studies showed that the kcat/Km values of the mutants K344R, H405R, and K344R/H405R under pH 4.5 were about 8-, 11.5-, and 17.7-times higher than that of the wild type enzyme. As revealed by the structure models of the wild-type and mutant enzymes, the amino acids substituted of R344 and R405 in the BLA contribute to its acid stability.

scholarly journals Substitution of His-181 by alanine in yeast phosphoglycerate mutase leads to cofactor-induced dissociation of the tetrameric structure

1993 ◽  
Vol 291 (2) ◽  
pp. 479-483 ◽  
Author(s):  
M F White ◽  
L A Fothergill-Gilmore ◽  
S M Kelly ◽  
N C Price
Keyword(s):  
Mutant Enzyme ◽  
Phosphoglycerate Mutase ◽  
Cross Linking ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Monomeric Species ◽  
Structure And Stability ◽  
Tetrameric Structure ◽  
Quaternary Structures ◽  
Tetrameric Form

The structure and stability of a mutated yeast phosphoglycerate mutase in which His-181 has been replaced by alanine have been studied. The secondary, tertiary and quaternary structures of the mutant enzyme in the absence of ligands are essentially identical to those of the wild-type enzyme as revealed by c.d., fluorescence and cross-linking studies. The mutant enzyme is slightly less stable than the wild-type enzyme towards denaturation by guanidium chloride (GdnHCl). On addition of cofactor 2,3-bisphosphoglycerate, the wild-type enzyme shows increased stability towards GdnHCl. However, addition of cofactor causes dramatic changes in the structure of the mutant enzyme, leading to dissociation of the tetrameric form to dimeric and monomeric species.

scholarly journals RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 968 ◽  
Author(s):  
Hyejeong Kim ◽  
Victor D. Ellis ◽  
Andrew Woodman ◽  
Yan Zhao ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Double Mutant ◽  
Biochemical Properties ◽  
Model System ◽  
Viral Population ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Rna Dependent Rna Polymerase ◽  
Or Efficiency ◽  
Coding Sequence

Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.

scholarly journals Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid

1997 ◽  
Vol 327 (2) ◽  
pp. 593-600 ◽  
Author(s):  
Li-Fan LIU ◽  
Yen-Chywan LIAW ◽  
F. Ming TAM
Keyword(s):  
Escherichia Coli ◽  
Free Energy ◽  
Binding Site ◽  
Gibbs Free Energy ◽  
Double Mutant ◽  
Ethacrynic Acid ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Wild Type Enzyme ◽  
Liver Glutathione

Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (ΔΔG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1. Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg). The kcat of this reaction can be increased 2.5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively. The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme. Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.

scholarly journals Effect of Metal Binding on the Structural Stability of Pigeon Liver Malic Enzyme

2001 ◽  
Vol 277 (7) ◽  
pp. 4663-4671 ◽  
Author(s):  
Hui-Chuan Chang ◽  
Wei-Yuan Chou ◽  
Gu-Gang Chang
Keyword(s):  
Structural Stability ◽  
Metal Binding ◽  
Malic Enzyme ◽  
Pigeon Liver
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