Conformational stability of pig citrate synthase and some active-site mutants.


Abstract

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme. Study holds ProTherm entries: 332, 333, 334, 335, 336, 337, 338 Extra Details:

Submission Details

ID: FA7e7ktr3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Zhi W;Srere PA;Evans CT,Biochemistry (1991) Conformational stability of pig citrate synthase and some active-site mutants. PMID:1892835
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
3NZI 2010-07-16T00:00:00+0000 2.75 Substrate induced remodeling of the active site regulates HtrA1 activity
5UZP 2017-02-27T00:00:00+0000 2.29 Crystal structure of citrate synthase mutant A348G from homo sapiens
5UZQ 2017-02-27T00:00:00+0000 2.16 Crystal structure of citrate synthase from homo sapiens
5UZR 2017-02-27T00:00:00+0000 2.3 Crystal structure of citrate synthase from homo sapiens
1CTS 1984-01-27T00:00:00+0000 2.7 CRYSTALLOGRAPHIC REFINEMENT AND ATOMIC MODELS OF TWO DIFFERENT FORMS OF CITRATE SYNTHASE AT 2.7 AND 1.7 ANGSTROMS RESOLUTION
2CTS 1984-01-27T00:00:00+0000 2.0 CRYSTALLOGRAPHIC REFINEMENT AND ATOMIC MODELS OF TWO DIFFERENT FORMS OF CITRATE SYNTHASE AT 2.7 AND 1.7 ANGSTROMS RESOLUTION
3ENJ 2008-09-25T00:00:00+0000 1.78 Structure of Pig Heart Citrate Synthase at 1.78 A resolution
4CTS 1984-01-27T00:00:00+0000 2.9 CRYSTAL STRUCTURE ANALYSIS AND MOLECULAR MODEL OF A COMPLEX OF CITRATE SYNTHASE WITH OXALOACETATE AND S-ACETONYL-COENZYME A
5UQS 2017-02-08T00:00:00+0000 1.6 Crystal structure of Citrate synthase from Sus scrofa
1AL6 1997-06-12T00:00:00+0000 1.85 CHICKEN CITRATE SYNTHASE COMPLEX WITH N-HYDROXYAMIDO-COA AND OXALOACETATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.2 Citrate synthase, mitochondrial Q0QEL7 CISY_MESAU
91.9 Citrate synthase, mitochondrial P23007 CISY_CHICK
95.9 Citrate synthase, mitochondrial Q8VHF5 CISY_RAT
96.3 Citrate synthase, mitochondrial O75390 CISY_HUMAN
96.1 Citrate synthase, mitochondrial Q9CZU6 CISY_MOUSE
96.8 Citrate synthase, mitochondrial P0C1Z2 CISY_MACFA
96.6 Citrate synthase, mitochondrial Q29RK1 CISY_BOVIN
100.0 Citrate synthase, mitochondrial P00889 CISY_PIG
90.4 Citrate synthase, mitochondrial Q6S9V6 CISY_XIPGL
90.2 Citrate synthase, mitochondrial Q6S9V8 CISY_THUOB
90.2 Citrate synthase, mitochondrial Q6S9V9 CISY_THUAL
90.2 Citrate synthase, mitochondrial Q6S9V7 CISY_KATPE
90.8 Citrate synthase, mitochondrial Q7ZWZ5 CISY_XENLA
91.8 Citrate synthase, mitochondrial Q28DK1 CISY_XENTR