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
1AG1 1997-03-28T00:00:00+0000 2.36 MONOHYDROGEN PHOSPHATE BINDING TO TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
1DKW 1999-12-08T00:00:00+0000 2.65 CRYSTAL STRUCTURE OF TRIOSE-PHOSPHATE ISOMERASE WITH MODIFIED SUBSTRATE BINDING SITE
1IIG 2001-04-23T00:00:00+0000 2.6 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHONOPROPIONATE
1IIH 2001-04-23T00:00:00+0000 2.2 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHOGLYCERATE
1KV5 2002-01-25T00:00:00+0000 1.65 Structure of Trypanosoma brucei brucei TIM with the salt-bridge-forming residue Arg191 mutated to Ser
1ML1 1996-09-27T00:00:00+0000 2.6 PROTEIN ENGINEERING WITH MONOMERIC TRIOSEPHOSPHATE ISOMERASE: THE MODELLING AND STRUCTURE VERIFICATION OF A SEVEN RESIDUE LOOP
1MSS 1994-07-27T00:00:00+0000 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
1TPD 1994-02-28T00:00:00+0000 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
1TPE 1994-02-28T00:00:00+0000 2.1 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
1TPF 1994-02-28T00:00:00+0000 1.8 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS

Relevant UniProtKB Entries

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