Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme.


Abstract

In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a k(cat) approximately 1000 times lower and a K(m) approximately 6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 A(3). The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis. Study holds ProTherm entries: 14876, 14877 Extra Details: 1 mM dithiothreitol and 1 mM azide were added in the experiments. dimer interface; activity; secondary structure; aromatic residues

Submission Details

ID: TsyuAfPp3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Hernández-Alcántara G;Garza-Ramos G;Hernández GM;Gómez-Puyou A;Pérez-Montfort R,Biochemistry (2002) Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme. PMID:11914068
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