A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability.


Abstract

Triosephosphate isomerase (TIM) is a very stable dimer. In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively. In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit). This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM. The monomeric RE-TIM has residual TIM activity. The thermal stability of RE-TIM is lower than that for wild type TIM. CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM. Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits. This implies that wild type monomeric subunits have some stability and are catalytically active. It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km. Study holds ProTherm entries: 3406, 3407 Extra Details: additive : EDTA(1 mM), Triosephosphate isomerase; TIM; double mutation;,thermal stability; flexible loops

Submission Details

ID: jqCd8JGG3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Schliebs W;Thanki N;Jaenicke R;Wierenga RK,Biochemistry (1997) A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability. PMID:9245397
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2J27 2007-01-02 1.15 The functional role of the conserved active site proline of triosephosphate isomerase
2V2H 2008-02-19 1.18 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
4PC8 2015-04-22 1.55 Structure-based protein engineering efforts on the scaffold of a monomeric triosephosphate isomerase yielding a sugar isomerase
2VEK 2008-02-19 1.6 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
1KV5 2002-03-29 1.65 Structure of Trypanosoma brucei brucei TIM with the salt-bridge-forming residue Arg191 mutated to Ser
3Q37 2011-09-14 1.65 Identification of Amino Acids that Account for Long-Range Interactions in Proteins Using Two Triosephosphate Isomerases from Pathogenic Trypanosomes.
2WSR 2009-09-15 1.65 MONOTIM MUTANT RMM0-1, MONOMERIC FORM.
5I3F 2016-05-18 1.72 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
1TPF 1994-05-31 1.8 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
5I3J 2016-05-18 1.8 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
5TIM 1992-10-15 1.83 REFINED 1.83 ANGSTROMS STRUCTURE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE, CRYSTALLIZED IN THE PRESENCE OF 2.4 M-AMMONIUM SULPHATE. A COMPARISON WITH THE STRUCTURE OF THE TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE-GLYCEROL-3-PHOSPHATE COMPLEX
2X1T 2010-01-26 1.83 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2X1U 2010-01-26 1.84 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2VEI 2008-02-19 1.89 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2V2C 2008-02-19 1.89 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
2X2G 2010-01-26 1.9 CRYSTALLOGRAPHIC BINDING STUDIES WITH AN ENGINEERED MONOMERIC VARIANT OF TRIOSEPHOSPHATE ISOMERASE
2X1S 2010-01-26 1.93 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
5I3G 2016-05-18 1.96 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
2X1R 2010-01-26 1.98 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2VEN 2008-02-19 2.0 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2WSQ 2009-09-15 2.1 MonoTIM mutant RMM0-1, dimeric form.
1TPE 1994-05-31 2.1 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
2J24 2007-01-02 2.1 The functional role of the conserved active site proline of triosephosphate isomerase
1TPD 1994-05-31 2.1 STRUCTURES OF THE 'OPEN' AND 'CLOSED' STATE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE, AS OBSERVED IN A NEW CRYSTAL FORM: IMPLICATIONS FOR THE REACTION MECHANISM
2X16 2009-12-29 2.13 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2V0T 2008-02-19 2.2 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
2VEM 2008-02-19 2.2 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
6TIM 1992-10-15 2.2 THE ADAPTABILITY OF THE ACTIVE SITE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE AS OBSERVED IN THE CRYSTAL STRUCTURES OF THREE DIFFERENT COMPLEXES
5I3I 2016-05-18 2.2 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
1IIH 2001-05-11 2.2 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHOGLYCERATE
5I3K 2016-05-18 2.21 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
5I3H 2016-05-18 2.25 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
2V2D 2008-02-19 2.3 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
4JEQ 2013-10-02 2.3 Different Contribution of Conserved Amino Acids to the Global Properties of Homologous Enzymes
2Y70 2011-12-07 2.3 CRYSTALLOGRAPHIC STRUCTURE OF GM23, MUTANT G89D, AN EXAMPLE OF CATALYTIC MIGRATION FROM TIM TO THIAMIN PHOSPHATE SYNTHASE.
2VEL 2008-02-19 2.3 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
1AG1 1997-06-16 2.36 MONOHYDROGEN PHOSPHATE BINDING TO TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
2V5L 2007-07-31 2.4 Structures of the Open and Closed State of Trypanosomal Triosephosphate Isomerase: as Observed in a New Crystal Form: Implications for the Reaction Mechanism
1TRI 1994-07-31 2.4 THE CRYSTAL STRUCTURE OF AN ENGINEERED MONOMERIC TRIOSEPHOSPHATE ISOMERASE, MONOTIM: THE CORRECT MODELLING OF AN EIGHT-RESIDUE LOOP
1TTI 1995-10-15 2.4 THREE NEW CRYSTAL STRUCTURES OF POINT MUTATION VARIANTS OF MONOTIM: CONFORMATIONAL FLEXIBILITY OF LOOP-1,LOOP-4 AND LOOP-8
1MSS 1994-09-30 2.4 LARGE SCALE STRUCTURAL REARRANGEMENTS OF THE FRONT LOOPS IN MONOMERISED TRIOSEPHOSPHATE ISOMERASE, AS DEDUCED FROM THE COMPARISON OF THE STRUCTURAL PROPERTIES OF MONOTIM AND ITS POINT MUTATION VARIANT MONOSS
4TIM 1992-10-15 2.4 CRYSTALLOGRAPHIC AND MOLECULAR MODELING STUDIES ON TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE: A CRITICAL ASSESSMENT OF THE PREDICTED AND OBSERVED STRUCTURES OF THE COMPLEX WITH 2-PHOSPHOGLYCERATE
1TTJ 1995-09-15 2.4 THREE NEW CRYSTAL STRUCTURES OF POINT MUTATION VARIANTS OF MONOTIM: CONFORMATIONAL FLEXIBILITY OF LOOP-1,LOOP-4 AND LOOP-8
1TRD 1993-10-31 2.5 THE INFLUENCE OF CRYSTAL PACKING ON CRYSTALLOGRAPHIC BINDING STUDIES: A NEW CRYSTAL FORM OF TRYPANOSOMAL TIM
1IIG 2001-05-11 2.6 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHONOPROPIONATE
2Y6Z 2011-12-07 2.6 Crystallographic structure of GM23 an example of Catalytic migration from TIM to thiamin phosphate synthase.
1ML1 1997-03-12 2.6 PROTEIN ENGINEERING WITH MONOMERIC TRIOSEPHOSPHATE ISOMERASE: THE MODELLING AND STRUCTURE VERIFICATION OF A SEVEN RESIDUE LOOP
1DKW 2000-11-03 2.65 CRYSTAL STRUCTURE OF TRIOSE-PHOSPHATE ISOMERASE WITH MODIFIED SUBSTRATE BINDING SITE
4PCF 2015-04-22 2.71 Structure-based protein engineering of a monomeric triosephosphate isomerase towards changing substrate specificity
3TIM 1991-10-15 2.8 THE CRYSTAL STRUCTURE OF THE 'OPEN' AND THE 'CLOSED' CONFORMATION OF THE FLEXIBLE LOOP OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
1TSI 1994-01-31 2.84 STRUCTURE OF THE COMPLEX BETWEEN TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE AND N-HYDROXY-4-PHOSPHONO-BUTANAMIDE: BINDING AT THE ACTIVE SITE DESPITE AN 'OPEN' FLEXIBLE LOOP

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Triosephosphate isomerase, glycosomal P04789 TPIS_TRYBB